Modulation of endothelial cell surface receptor activity in the regulation of angiogenesis

ABSTRACT

A method of modulating angiogenesis in a vertebrate subject, the method comprising administering to the vertebrate subject an ECRTP/DEP-1 activity-modulating amount of a composition, whereby an ECRTP/DEP-1 within the vertebrate subject is contacted by the composition; and modulating angiogenesis through the contacting of the ECRTP/DEP-1 with the composition. Optionally, the composition includes a monoclonal antibody which preferentially binds ECRTP/DEP-1. Methods for screening for modulators of ECRTP/DEP-1 are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/516,728, filed Mar. 1, 2000, which is a continuation-in-part ofco-pending U.S. patent application Ser. No. 09/152,160, filed Sep. 10,1998, now U.S. Pat. No. 6,248,327, the disclosure of each of which isherein incorporated by reference in its entirety.

GRANT STATEMENT

This work was supported by NIH grants DK38517 and CA 68485. Thus, theU.S. Government has certain rights in the invention.

TECHNICAL FIELD

The present invention relates generally to the modulation of theactivity of an endothelial cell surface receptor in the regulation ofendothelial cell proliferation and migration and in the regulation ofangiogenesis. More particularly, the present invention relates to themodulation of ECRTP/DEP-1 activity in the regulation of endothelial cellproliferation and migration and in the regulation of angiogenesis.

BACKGROUND

As used herein, the term “angiogenesis” means the generation of newblood vessels into a tissue or organ. Under normal physiologicalconditions, humans or animals undergo angiogenesis only in very specificrestricted situations. For example, angiogenesis is normally observed inwound healing, fetal and embryonal development and formation of thecorpus luteum, endometrium and placenta. The term “endothelium” means athin layer of flat epithelial cells that lines serous cavities, lymphvessels, and blood vessels. The term “endothelial modulating activity”means the capability of a molecule to modulate angiogenesis in generaland, for example, to stimulate or inhibit the growth of endothelialcells in culture. Both controlled and uncontrolled angiogenesis arethought to proceed in a similar manner. Endothelial cells and pericytes,surrounded by a basement membrane, form capillary blood vessels.Angiogenesis begins with the erosion of the basement membrane by enzymesreleased by endothelial cells and leukocytes. The endothelial cells,which line the lumen of blood vessels, then protrude through thebasement membrane. Angiogenic stimulants induce the endothelial cells tomigrate through the eroded basement membrane. The migrating cells form a“sprout” off the parent blood vessel, where the endothelial cellsundergo mitosis and proliferate. The endothelial sprouts merge with eachother to form capillary loops, creating the new blood vessel.

Persistent, unregulated angiogenesis occurs in a multiplicity of diseasestates, and abnormal growth by endothelial cells and supports thepathological damage seen in these conditions. The diverse pathologicaldisease states in which unregulated angiogenesis is present have beengrouped together as angiogenic dependent or angiogenic associateddiseases.

It is also recognized that angiogenesis plays a major role in themetastasis of a cancer. If this angiogenic activity could be repressedor eliminated, then the tumor, although present, would not grow. In thedisease state, prevention of angiogenesis could avert the damage causedby the invasion of the new microvascular system. Therapies directed atcontrol of the angiogenic processes could lead to the abrogation ormitigation of these diseases.

The development of renal glomerular capillaries is anatomicallysegregated and temporally staged in a multi-step process. The processinvolves recruitment of endothelial progenitors from adjacentmesenchyme, assembly of an arborized branching network, and maturationand specialization of endothelial cells adjacent to mesangial andvisceral epithelial cells. Receptors for extracellular matrixcomponents, cell surface molecules and growth factors have been assignedroles to mediate steps in this assembly process. See e.g., Wallner etal., Microsc Res Tech 39:261-284 (1997); Takahashi et al., Kidney Int53:826-835 (1998).

Vascular endothelial growth factor (VEGF) is an important participant,as it is induced in S stage developing glomerular epithelial cells, andendothelial progenitors that are recruited to glomerular capillariesfrom the adjacent metanephric mesenchyme express the VEGF receptor,flk-1. Robert et al., Am J Physiol 271:F744-F753 (1996).

Neutralizing VEGF antibodies interrupt postnatal murine glomerularcapillary development. Kitamoto et al., J Clin Invest 99:2351-2357(1997). Deletion of either PDGFβ receptor or PDGFβ genes in mice causesdefective recruitment of mesangial cell precursors with failure ofglomerular development. Soriano, P., Genes Dev 8:1888-1896 (1994);Leveen et al., Genes Dev 8:1875-1887 (1994). TGFβ1 expression and typeII TGFβ receptors appear critical for vascular development in theembryonic yolk sac (prior to renal development), and type II receptorsmediate in vitro capillary morphogenesis of endothelial cells derivedfrom bovine glomeruli. Choime et al., J Biol Chem 270:21144-21150(1995).

Early evidence suggests that Eph family receptors and their ephrinligands participate in glomerular vascular development. EphB1 receptorsare expressed in isolated mesenchymal cells in a pattern similar to thatof flk-1, and high level expression of ephrin-B1 is seen at the vascularcleft of developing glomeruli, as well as in capillary endothelial cellsof mature glomeruli. Daniel et al., Kidney Int 50:S-73-S-81 (1996).Oligomerized forms of ephrin-B1 stimulate in vitro assembly of humanrenal microvascular endothelial cells (HRMEC) into capillary-likestructures. Stein et al., Genes Dev 12:667-678 (1998).

A selected subclass of receptor tyrosine phosphatases, includingDPTP10D, serve important roles in directing axonal migration and neuralnetwork assembly. Desai et al., Cell 84:599-609 (1996). Recent data hasidentified mRNA expression of a related receptor phosphatase,ECRTP/DEP-1, in arterial sites in mammalian kidney. Borges et al.,Circulation Research 79:570-580 (1996). To date, however, there has beenno evidence to implicate receptor tyrosine phosphatases in microvascularor glomerular capillary assembly or maturation.

Vascular endothelial cells display a diverse range of vascular bedspecific properties (Gumkowski et al., Blood Vessels 24:11-13 (1987)),yet the requirement to maintain a continuous, antithrombotic monolayerlining the vascular space imposes rigorous requirements that theirproliferation, migration and differentiation be regulated byinterendothelial contacts. Specialized intercellular contacts permitcommunication among interacting endothelial cells (Lampugnani et al., JCell Biol 129:203-217 (1995)) yet the mechanisms regulating arrest ofproliferation and migration in response to interendothelial contact havenot been elucidated. Tight regulatory control over proliferation imposedby interendothelial cell contact is apparent in the low basal mitoticindex among endothelial cells in existing vessels. Engerman et al.,Laboratory Investigation 17:738-744 (1967). This is in contrast with theproliferative endothelial responses that are evoked by mechanicaldisruption of large vessels. More et al., J Pathol 172:287-292 (1994).Similar proliferation and migration responses are stimulated at themargin of a confluent endothelial monolayer by “wounding”, or physicalremoval cells from the packed monolayer. Coomber, J Cell Biochem52:289-296 (1993).

The molecular basis for effects of interendothelial contact on migratoryand proliferative responses is not defined, yet studies of culturedcells have shown that endothelial, fibroblast, and epithelial cells growto confluency at a predictable density, then arrest proliferation(density arrest). Augenlicht and Baserga, Exp Cell Res 89:255-262(1974); Beekhuizen and van Furth, J Vascular Res 31:230-239 (1994);Rijksen et al., J Cell Physiol 154:393-401 (1993). This phenomenon canbe very relevant to the behavior of endothelial cells in vascular sitesin situ. Indeed, model culture systems of endothelial “wounding” haveshown that endothelial cells at the edge of an imposed “wound” rapidlyextend lamellae, spread, migrate and proliferate to replace the deficitcreated by mechanical disruption of the monolayer. Coomber, J CellBiochem 52:289-296 (1993).

Pallen and Tong observed that membrane-associated tyrosine phosphataseactivity recovered from cultured Swiss 3T3 cells increased eight(8)-fold (expressed as activity/mg protein) as cells approached adensity of 5×10⁴/cm², while soluble fraction tyrosine phosphatase wasunaffected by cell density. Pallen and Tong, Proc Natl Acad Sci USA88:6996-7000 (1991). Ostman et al. determined that the abundance of areceptor tyrosine phosphatase cloned from HeLa cells and named DEP-1, isincreased as cells approach high density. Ostman et al., Proc Natl AcadSci USA 91:9680-9684 (1994). However, no links between molecules thatevoke proliferation arrest and receptor tyrosine phosphatases have beenmade.

To date, available information does not indicate what sort ofreceptor-ligand interaction might mediate a cell surface generatedsignal for density or contact arrest. The identification of such areceptor-ligand interaction is therefore needed in that it will serve asa basis for intervention in a disorder wherein density or contactarrest, or the preclusion of density or contact arrest, has therapeuticvalue. Such disorders include disorders characterized by undesiredangiogenesis, such as angiogenesis associated with tumor growth. Thus,what is also needed is a composition and method which can inhibit theunwanted growth of blood vessels, especially into tumors. Thecomposition and method should attenuate the formation of the capillariesin the tumors thereby inhibiting the growth of the tumors.

SUMMARY

In accordance with the present invention, a method of modulatingangiogenesis in a vertebrate subject is provided. The method comprisesadministering to the vertebrate subject an ECRTP/DEP-1 activitymodulating amount of a composition, whereby an ECRTP/DEP-1 within thevertebrate subject is contacted by the composition; and modulatingangiogenesis through the contacting of the ECRTP/DEP-1 with thecomposition.

In accordance with the present invention a method of modulatingendothelial cell migration and proliferation in a vertebrate subject isalso provided. The method comprises administering to the vertebratesubject an ECRTP/DEP-1 activity-modulating amount of a composition,whereby an ECRTP/DEP-1 within the vertebrate subject is contacted by thecomposition; and modulating endothelial cell migration and proliferationthrough the contacting of the ECRTP/DEP-1 with the composition.

In accordance with the present invention there is also provided anantibody which preferentially binds the ECRTP/DEP-1. Optionally, theantibody comprises a monoclonal antibody or fragment or derivativethereof which preferentially binds the ECRTP/DEP-1.

In accordance with the present invention, a method for isolating anendogenous ligand for an ECRTP/DEP-1 is also provided. The methodcomprises the steps of contacting cells or cell lysates having theligand with ECRTP/DEP-1; and isolating the ligand which binds withECRTP/DEP-1.

A method of screening candidate substances for an ability to modulateECRTP/DEP-1 biological activity is also disclosed. The method comprisesestablishing test samples comprising an ECRTP/DEP-1 polypeptide orfragment thereof; administering a candidate substance to the testsamples; and measuring the interaction, effect, or combination thereof,of the candidate substance on the test sample to thereby determine theability of the candidate substance to modulate ECRTP/DEP-1 biologicalactivity.

In accordance with the present invention there are also provided methodsfor performing a screening assay for identifying a compound thatmodulates an activity of an ECRTP/DEP-1 in both a cell-based and acell-free assay. In a cell-based assay, the method comprises the stepsof establishing replicate test and control cultures of cells thatexpress the ECRTP/DEP-1; administering a candidate compound to the cellsin the test culture but not the control culture; measuring ECRTP/DEP-1activity in cells in the test and the control cultures; and determiningthat the candidate compound modulates the ECRTP/DEP-1 activity in a cellif the ECRTP/DEP-1 activity measured for the test culture is greater orless than the ECRTP/DEP-1 activity measured for the control culture.

In a cell-free system, the method comprises the steps of establishing acontrol system comprising an ECRTP/DEP-1 and a ligand wherein theECRTP/DEP-1 is capable of binding to the ligand; establishing a testsystem comprising the ECRTP/DEP-1, the ligand, and a candidate compound;measuring the binding affinity of the ECRTP/DEP-1 and the ligand in thecontrol and the test systems; and determining that the candidatecompound modulates ECRTP/DEP-1 activity in a cell-free system if thebinding affinity measured for the test system is less than or greaterthan the binding affinity measured for the control system.

In another embodiment, the screening assay methods of the presentinvention pertain to comparing the effect of a candidate compound toinhibit growth of cells expressing exogenous ECRTP/DEP-1 compared withthose not expressing ECRTP/DEP-1 and determining that the effect ofaltering ECRTP/DEP-1 activity is responsible by demonstrating the lackof activity of the candidate compound on cells not expressingECRTP/DEP-1. Thus, the screening assays of the present invention showchanges in growth that are responsive to changes in ECRTP/DEP-1activity.

In accordance with the present invention there is also provided a methodfor delivering a therapeutic composition to a tissue in a patient,wherein the tissue is characterized as having undesirable endothelialcell proliferation. The method comprises the steps of introducing intothe patient a biologically effective amount of an antibody operativelylinked to a selected therapeutic agent, the antibody preferentiallybinding to an ECRTP/DEP-1 on the surface of the endothelial cells,whereby an ECRTP/DEP-1 within the vertebrate subject is contacted by theantibody; and delivering the therapeutic composition to the tissuethrough the contacting of the ECRTP/DEP-1 with the composition.

It is therefore an object of the present invention to localize andcharacterize a receptor-ligand interaction which mediates a cellsurface-generated signal for cell growth and survival.

It is another object of the present invention to provide for themodulation of a cell surface receptor activity in endothelial cells tomediate a cell surface-generated signal for cell growth and survival.

It is still another object of the present invention to provide for themodulation of a cell surface receptor activity for use in the inhibitionor stimulation of angiogenesis.

It is yet another object of the present invention to identify compoundswhich modulate a receptor-ligand interaction which mediates a cellsurface-generated signal for density or contact arrest.

Some of the aspects and objects of the invention having been statedhereinabove, other aspects and objects will become evident as thedescription proceeds, when taken in connection with the accompanyingDrawings and Examples as best described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts recognition by antibodies ECRTPAb-1 & ECRTPAb-2 ofrecombinant and over-expressed ECRTP/DEP-1 and is an autoradiographdepicting recombinant proteins representing extracellular (Ec) orcytoplasmic (Cy) domains of ECRTP/DEP-1 were expressed in bacteria andpurified. Proteins (100 ng) were separated on a 15% SDS-polyacrylamidegels, transferred to PVDF membrane and probed with monoclonal antibodiesECRTPAb-1 or ECRTPAb-2, as indicated.

FIG. 1B depicts recognition by antibodies ECRTPAb-1 & ECRTPAb-2 ofrecombinant and over-expressed ECRTP/DEP-1 and is an autoradiographdepicting MDCK cells cultured in 100 mm dishes were transfected with 14μg of empty pSRα vector (SRa) or pSRα-ECRTP/DEP-1/HA (SRa-ECRTP/HA)expression constructs and harvested at 48 hours after transfection.Membrane receptor proteins were recovered by WGA lectin-conjugatedagarose from 150 μg of lysate protein. Lectin-adsorbed, eluted proteinswere subjected to 7% SDS-PAGE, transferred to a PVDF membrane and probedwith ECRTPAb-1, ECRTPAb-2, or anti-HA (HAAb) monoclonal antibodies, asindicated.

FIG. 1C is a series of photographs depicting MDCK cells stablytransfected with the pSR_-ECRTP/DEP-1/HA plasmid were fixed with coldmethanol and stained with ECRTPAb-2 (panels a, c & d) or a class matchedcontrol antibody (panel b). ECRTPAb-2 labeled lateral borders of cellsin contact. Preincubation of ECRTPAb-2 with 50 μg of recombinantimmunogen (Ec) blocked this staining (panel c), while an irrelevantrecombinant protein (Cy) did not (panel d).

FIGS. 2A-2E are a series of photographs depicting the abundance ofECRTP/DEP-1 in endothelial cells of adult human kidney. Acetone fixedfrozen sections (5 μm thickness) of human kidney were incubated withECRTPAb-1 (panels A-D) or a class matched control monoclonal antibody(panel E) and bound antibody was detected by epifluorescence microscopy,as described in Methods. ECRTPAb-1 prominently labeled glomerular,peritubular and arterial endothelial cells. Magnifications were A)×100;B)×600; C)×600; D×400; and E)×100.

FIG. 3 depicts confocal localization of ECRTP/DEP-1 and VE cadherin inhuman kidney vasculature. Acetone fixed kidney sections weresimultaneously labeled with ECRTPAb-1 and a polyclonal goat antibodyagainst VE cadherin. Bound antibodies were detected using fluoresceinconjungated anti-mouse (panels A, B, E, F) or rhodamine-conjugatedanti-goat (panels C, D, E, F) Ig antibodies. ECRTPAb-1 (green) stainingdistributed over the entire endothelial membrane in large artery andglomerular capillaries (A, B) while VE cadherin labeling (red) isrestricted to endothelial junctions (C, D). Overlapping confocal imagesdemonstrated colocalization of ECRTP with VE cadherin atinter-endothelial junctions. (magnification×600).

FIG. 4 is a series of photographs depicting ECRTP/DEP-1 expression indeveloping murine glomeruli. Cryostat kidney sections of embryonic day14 (A), day 16 (B), postnatal day 6 (C) and adult mice (D) wereimmunolabeled with ECRTPAb-1 as described in the Methods of Example 1.In panels A & B; ECRTPAb-1 binds to cells dispersed in the mesenchymalarea (arrow), to endothelial precursor cells (arrowhead) migrating tothe vascular cleft of comma-shaped glomeruli and to endothelium ofcapillary stage glomeruli (G). In panels C & D, ECRTPAb-1 preferentiallylabels endothelial cells of the glomerulus (G), artery (A) andperitubular capillaries (arrow) in mature kidney. (Originalmagnification; A)×400; B)×200; C)×200; and D)×350.

FIG. 5A depicts distribution of ECRTP/DEP-1 of inter-endothelialcontacts in cultured human endothelial cells, but ECRTP/DEP-1 does notdissociate from junctions with VE cadherin and is a series ofphotographs depicting Methanol fixed HRMEC cells were labeled withECRTPAb-2 as described in Methods of Example 1. ECRTP/DEP-1 isdistributed between points of inter-endothelial membrane contact andpunctate regions of the apical membrane in serial confocal images.

FIG. 5B depicts distribution of ECRTP/DEP-1 of inter-endothelialcontacts in cultured human endothelial cells, but ECRTP/DEP-1 does notdissociate from junctions with VE cadherin and is a series ofphotographs depicting HMEC-1 cells were grown to confluency, thenincubated with media containing 5 mM EGTA for 0 min (panels a & c) or 20min (panels b & d), prior to fixation. The distribution of ECRTPAb-2 andVE cadherin labeling was examined as described in Methods of Example 1at each time. While the distribution of ECRTP/DEP-1 immunoreactivity wasnot altered in the low Ca²⁺ medium, junctional VE cadherin stainingdissipated, consistent with dissociation of VE cadherin junctions andredistribution across the cell membrane.

FIG. 6A shows that endothelial cell density imposes growth arrest andincreases lectin recoverable tyrosine phosphatase activity and is a linegraph showing identical numbers of human renal microvascular endothelialcells (HRMEC) were plated in growth medium on 100 (1×), 60 (2.9×) or 35(8.1×) mm diameter plastic dishes, effecting the indicated folddifferences in cell density at the time of plating. Medium was replacedwith growth medium at points indicated by arrows. Cells were counted ina Coulter counter and means of quadruplicate samples are displayed.Proliferation was arrested in cells at 8.1× density after a single celldoubling, and after approximately 3 doublings in cells plated at 2.9×density.

FIG. 6B shows that endothelial cell density imposes growth arrest andincreases lectin recoverable tyrosine phosphatase activity and is a bargraph showing cells plated for the indicated times at the indicateddensities were lysed, and receptor tyrosine phosphatase activity,including that attributable to ECRTP/DEP-1, was recovered by lectinaffinity chromatography and assayed as described in Methods of Example 2in the absence or presence of the tyrosine phosphatase inhibitor, sodiumorthovanadate (VO₄, 100 μM).

FIG. 7 is an autoradiograph and a bar graph showing that increased celldensity imposes increases in activity, but not amount, ofimmunoprecipitated ECRTP/DEP-1. Identical numbers of HRMEC were platedas in FIG. 6 at the indicated cell densities. Monospecific affinitypurified rabbit polyclonal antibodies were used to immunoprecipitateECRTP/DEP-1 from cells treated for 10 min immediately before harvestwith 1 mM peroxyvanadate (+VO₄) or vehicle (−VO₄) at 36 hours afterplating, as described in Methods of Example 2. Recovered ECRTP/DEP-1antigen was quantitated by immunoblot with the monospecific antibody andits endogenous phosphotyrosine content assessed by phosphotyrosineimmunoblot using the 4G10 monoclonal antibody. Phosphatase activity inimmunoprecipitated samples was assayed using pNPP as substrate in theabsence (−VO₄) or presence (VO₄) of sodium orthovanadate, as described.Data are displayed as optical density of the product in triplicatesamples +/−SEM.

FIG. 8A shows that ECRTP/DEP-1 overexpression, or bivalent antibodyagainst ECRTP/DEP-1, ECRTPAb-1, imposes proliferation arrest on HRMECand is a graph showing transient transfection of HMREC with ECRTP/DEP-1cDNA imposes a growth inhibition at low cell densities. Approximately3×10⁵ HRMEC were cotransfected with 1.7 μg pSRα (vector control) or HAepitope tagged (hemagglutinin) pSRα-ECRTP/DEP-1 (pSRα-ECRTP), asindicated, and 0.4 μg pEGFP (Clontech) to permit scoring of BrdUlabeling of transfected cells, as described in Methods of Example 2. At24 hours, transfected cells were replated on p35 dishes in the numbersindicated. Thirty six hours later, S phase cells were labeled for 30 minwith BrdU, as described in Methods of Example 2, and +GFP positive cellswere scored for BrdU incorporation. Data represent means+/−SEM forquadruplicate determinations.

FIG. 8B shows that ECRTP/DEP-1 overexpression, or bivalent antibodyagainst ECRTP/DEP-1, ECRTPAb-1, imposes proliferation arrest on HRMECand is a line graph showing that ECRTPAb-1 inhibits endothelialproliferation and migration. HRMEC (3×10⁴) were plated in p35 dishes attime 0. Growth medium was replaced at 24 h, cells were counted, andeither IgG control (10 μg/ml) or ECRTPAb1 (10 μg/ml) antibodies wereadded. Replicate samples (5) of cells were counted on day 4, and areexpressed as means+/−SEM.

FIG. 8C is a data point plot depicting that equal numbers of HRMEC wereplated at time 0, and antibodies or Fab fragments added at theconcentrations indicated. Replicate plates were harvested on day 1, toconfirm homogeneous plating efficiency in each condition, and on day 6to assess cell proliferation, respectively. Data points represent meanvalues of five replicates ±SEM.

FIG. 9A depicts inhibition of endothelial migration by ECRTPAb-1 and isa series of photographs depicting monolayers of HRMEC were transientlytransfected with plasmid pSRαECRTP/DEP-1/HA, or pSRαEphB1/HA, asindicated. Forty eight hours later, “wounds” were created in theconfluent monolayers and permitted to close over the ensuing 30 h.Monolayers were then stained with the monoclonal hemagglutinin antibody,12CA5, to detect the positions of cells transiently expressing highlevels of ECRTP/DEP-1/HA or EphB1/HA, respectively. Only rareECRTP/DEP-1 overexpressing cells migrated to close the “wound”.

FIG. 9B depicts inhibition of endothelial migration by ECRTPAb-1 and isa line graph reflecting analysis of 300 to 420 μm diameter “wounds”which were created in HRMEC confluent monolayers at time 0, as mediumwas exchanged to serum-free medium supplemented by either no addition(NA), or phorbol myristate acetate (20 ng/ml) in the presence of theindicated antibodies or fragments, including a class matched IgG control(IgG, 10 μg/ml), ECRTPAb1 (10 μg/ml), or Fab fragments of ECRTPAb1 (3μg/ml, molar equivalency). Triplicate wounds were used to generatemicroscopic images at the indicated times, and the residual “wound” areacalculated and expressed as a fraction of the original wound, by anautomated capture sequence using Bioquant Image Analysis Software. Eachdata point represents the mean±SEM of three determinations.

FIG. 9C depicts inhibition of endothelial migration by ECRTPAb-1 and isa line graph analyzing data produced by the same assay procedure as FIG.9B. Using the same assay procedure, migration rates were calculated bylinear regression of mean values determined in cells exposed to IgGcontrol, ECRTPAb1, or ECRTPAb1/Fab, using three independent time points.r² values≧0.90 for each data point plotted. The open square (□)indicates the migration rate for closure of unstimulated cells.

FIG. 10 is line graph depicting that ECRPTAb1 Fab fragments attenuateendothelial density mediated growth arrest. HMEC-1 cells of theindicated numbers were plated in on coverslips in 12 well dishes at time0 in growth media supplemented by no addition (NA) or ECRTPAb1 (67 nM).Twenty four hours later BrdU staining was assayed as described inMethods of Example 2 and the percentage of BrdU positive cells scored bycounting of five independent fields for each condition (greater than 400cells/point). Data represent means±SEM.

FIG. 11 is a series of photographs depicting that ECRTPAb1 inhibitscorneal pocket angiogenesis responses to bFGF. Hydron pellets wereimpregnated with the angiogenesis stimulant, basic FGF (90 ng), alone,or supplemented with a class matched control monoclonal antibody (IgG,200 ng) or ECRTPAb1 (200 ng), and placed in a pocket created in thecorneal epithelium of anesthetized mice. Five days after implantation,angiogenic responses were scored, and photographed. Representativeexamples show inclusion of the ECRTPAb1 inhibits the zone ofproliferation around the implanted pellet.

FIG. 12 is an autoradiograph showing that ECRTPAb-1 binds peptidesequence QSRDTEVL (SEQ ID NO: 1) of ECRTP/DEP-1 ectodomain. A “peptideson paper” (Research Genetics Inc., Huntsville, Ala.) array was generatedusing the 351 amino acid sequence of ECRTP which ECRTPAb-1 binds. Thearray comprises 96 peptides of 8-10 amino acids spanning the entireregion in overlapping sequences. A single peptide sequence (#41) in theseries represents amino acid residues in n-QSRDTEVL-c (SEQ ID NO: 1).

FIG. 13A is a combination of autoradiographs and a graph depicting thatECRTPAB-1 promotes dephosphorylation of ECRTP and arrests growth oftransfected CHO cells expressing wild type ECRTP, but not mutant ECRTPproteins, C/S (catalytically inactivated point mutant) or cy (acytoplasmic domain deletion). CHO cells were replated at 24 hours in thepresence of ECRTPAb-1, ECRTPAb-1-Fab, or control IgG1 and assayed forcell number at 48 hours after transfection. In right panels, cells weretransfected, cultured for 48 hours in serum-containing medium (5%), andexposed to 70 nM of ECRTPAb-1 or ECRTPAb-1-Fab for the times indicated.ECRTP was immunoprecipitated and assayed by immunoblot forphosphotyrosine content (anti-PY) and antigen recovery (anti-HA), asindicated. ECRTPAb-1, but not ECRTPAb-1-Fab, promoted acutedephosphorylation of wt but not catalytically inactive ECRTP.

FIG. 13B is a combination of autoradiographs and a graph demonstratingthat cotransfection of wild type ECRTP with either C/S or cy formsabrogates the dephosphorylation of ECRTP imposed by exposure of cells toECRTPAb-1. These mutant forms function as dominant negative proteins toblock the ECRTPAb-1-induced formation of catalytically active ECRTPdimers that arrest cell growth and promote ECRTP dephosphorylation.

DETAILED DESCRIPTION

A mammalian transmembrane protein gene product called DEP-1 (for densityenhanced phosphatase), ECRTP, PTPRJ, HPTPρ, CD148, BYP, depending uponspecies and cDNA origin), was initially cloned from fibroblasts and wassubsequently shown to be expressed (hereinafter referred to as an“ECRTP/DEP-1”) on all hematopoietic lineages (de la Fuente-Garcia etal., Blood 91:2800-2809 (1998), including erythroid progenitor cells,megakaryocytes and platelets, lymphocytes, polymorphononuclearleukocytes and platelets, and very prominently in endothelial cells.Borges et al., Circulation Research 79:570-580 (1996), Schoecklmann etal., J Am Soc Nephrol 5:730 (1994)(abstract). This gene product has beenshown to promote differentiation of erythroid progenitor cells (Kumet etal., J Biol Chem 271:30916-30921 (1996)), to modulate lymphocytefunction when crosslinked with other signaling proteins (de laFuente-Garcia et al., Blood 91:2800-2809 (1998)); and to inhibit clonalexpression of breast cancer cell lines overexpressing the protein (Keaneet al., Cancer Research 56:4236-4243 (1996)).

In accordance with the present invention, it has been demonstrated thatantibodies specific for ectodomain epitopes of the ECRTP/DEP-1 blockendothelial migration and proliferation in response to phorbol myristateacetate and fetal bovine serum respectively. It is recognized that thebiological activity to inhibit endothelial proliferation and migrationis a strong indicator of angiogenesis inhibitory activity. Accordingly,the ECRTP/DEP-1 is also a mediator of inhibitory signals that blockangiogenesis.

In accordance with the present invention, then, antibodies thataggregate the ECRTP/DEP-1, including monoclonal antibody ECRTPAb-1described herein, inhibit angiogenesis. Indeed, monoclonal antibodiesagainst the ectodomain of ECRTP/DEP-1 inhibit proliferation (asdemonstrated by BrdU uptake experiments) and migration of endothelialcells. Fab fragments of the same monoclonal have no such activity.Accordingly, such monoclonal ECRTP/DEP-1 antibodies described herein andderivatives thereof, have biological activity as angiogenesisinhibitors.

An endogenous ligand for the receptor ectodomain signals endothelialgrowth arrest. Therefore, in accordance with the present invention, amethod of screening for the endogenous ligand is provided. For example,the endogenous ligand is isolated through the preparation of fusionproteins of the ECRTP/DEP-1 ectodomain as affinity reagents to identify,establish assays for, and clone the putative natural ligand expressed onendothelial cells. The purified and isolated endogenous ligand thus alsohas therapeutic application as an angiogenesis inhibitor.

In accordance with the present invention, synthetic peptides andpeptidomimetics can also be used to contact the ECRTP/DEP-1 to activateECRTP/DEP-1 activity.

The ECRTP/DEP-1 is expressed on the luminal and interendothelialmembranes of endothelial cells in microvascular and large arterialvessels of kidney and other organs, including but not limited to heart,spleen, muscle and skin. The ECRTP/DEP-1 localizes to interendothelialcontacts in cultured endothelial cells, and in regions that overlap, butlocalization is not limited to the VE cadherin rich junctionalcomplexes. ECRTP/DEP-1 activity (tyrosine phosphatase activity)increases approximately two times in confluent cells anticipatingdensity mediated growth arrest. Moreover, over-expression of ECRTP/DEP-1confers growth arrest on subconfluent endothelial cells. Thus, inaccordance with the present invention, a method of modulatingECRTP/DEP-1 activity by contacting an ECRTP/DEP-1 with an ECRTP/DEP-1modulating composition is contemplated. A method of screening for such acomposition is also contemplated. Finally, a method of targeting atherapeutic composition to interendothelial contacts by preparing anantibody which preferably binds the ECRTP/DEP-1 and which is bound tothe therapeutic composition in provided in accordance with the presentinvention.

A. General Considerations

The present invention relates generally to the discovery thatangiogenesis is modulated by the ECRTP/DEP-1 and that activation ofECRTP/DEP-1 function inhibits angiogenesis. This discovery is importantbecause of the role that angiogenesis plays in a variety of diseaseprocesses. By modulating angiogenesis, one can intervene in the disease,ameliorate the symptoms, and in some cases cure the disease.

Where the growth of new blood vessels is the cause of, or contributesto, the pathology associated with a disease, inhibition of angiogenesiswill reduce the deleterious effects of the disease. Examples includerheumatoid arthritis, diabetic retinopathy, and the like. Where thegrowth of new blood vessels is required to support growth of adeleterious tissue, inhibition of angiogenesis will reduce the bloodsupply to the tissue and thereby contribute to reduction in tissue massbased on blood supply requirements. Examples include growth of tumorswhere neovascularization is a continual requirement in order that thetumor grow beyond a few millimeters in thickness, and for theestablishment of solid tumor metastases.

The methods of the present invention are effective in part because thetherapy is highly selective for angiogenesis and not other biologicalprocesses. As shown in the Examples, the ECRTP/DEP-1 localizes toendothelial cells and thus, primarily new vessel growth containssubstantial ECRTP/DEP-1, and therefore the therapeutic methods do notadversely affect mature vessels. Furthermore, the ECRTP/DEP-1 is notwidely distributed in normal tissues, but rather is found selectively onthe surface of endothelial cells, thereby assuring that the therapy canbe selectively targeted.

The discovery that binding the ECRTP/DEP-1 will effectively inhibitangiogenesis allows for the development of therapeutic compositions withpotentially high specificity, and therefore relatively low toxicity.Thus although the invention discloses the preferred use of ananti-ECRTP/DEP-1 monoclonal antibody, one can design reagents whichselectively bind ECRTP/DEP-1, and therefore do not have the side effectof modulating other biological processes other that those mediated byECRTP/DEP-1.

As shown by the present teachings, it is possible to prepare monoclonalantibodies highly selective for immunoreaction with the ECRTP/DEP-1 thatare similarly selective for modulation of ECRTP/DEP-1 function. Inaddition, peptides can be designed to be selective for binding toECRTP/DEP-1, as described further herein. Prior to the discoveries ofthe present invention, it was not known that angiogenesis could bemodulated in vivo by the use of reagents that modulate the biologicalfunction of ECRTP/DEP-1 or other receptor tyrosine phosphatase.

Other related methods are described in U.S. Pat. Nos. 5,753,230;5,733,876; 5,762,918; 5,776,427; 5,766,591; and 5,660,827, the entirecontents of each of which are herein incorporated by reference.

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

B. Methods for Modulating Angiogenesis

The invention provides for a method for the modulation of angiogenesisin a tissue, and thereby modulating events in the tissue which dependupon angiogenesis. Generally, the method comprises administering to thetissue a composition comprising an angiogenesis-modulating amount of anECRTP/DEP-1 modulator. As disclosed herein the term “modulate” is meantto encompass the inhibition or stimulation of angiogenesis. Thus, thetherapeutic methods of the present invention pertain to the inhibitionor stimulation of angiogenesis, depending on the disorder to be treated.

Angiogenesis includes a variety of processes involvingneovascularization of a tissue including “sprouting”, vasculogenesis, orvessel enlargement, all of which angiogenesis processes are mediated byand dependent upon the expression of ECRTP/DEP-1. With the exception oftraumatic wound healing, corpus luteum formation and embryogenesis, itis believed that many angiogenesis processes are associated with diseaseprocesses.

There are a variety of diseases in which angiogenesis is believed to beimportant, referred to as angiogenic diseases, including but not limitedto, inflammatory disorders such as immune and non-immune inflammation,chronic articular rheumatism and psoriasis, disorders associated withinappropriate or inopportune invasion of vessels such as diabeticretinopathy, neovascular glaucoma, capillary proliferation inatherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas, Karposi's sarcoma and the likecancers which require neovascularization to support tumor growth.

Thus, methods which inhibit angiogenesis in a diseased tissueameliorates symptoms of the disease and, depending upon the disease, cancontribute to cure of the disease. In one embodiment, the inventionpertains to inhibition of angiogenesis, per se, in a tissue. The extentof angiogenesis in a tissue, and therefore the extent of inhibitionachieved by the present methods, can be evaluated by a variety ofmethods, such as are described in the Examples for detecting anECRTP/DEP-1-immunopositive immature and nascent vessel structures byimmunohistochemistry.

As described herein, any of a variety of tissues, or organs comprised oforganized tissues, can support angiogenesis in disease conditionsincluding skin, muscle, gut, connective tissue, joints, bones and thelike tissue in which blood vessels can invade upon angiogenic stimuli.

Thus, in one related embodiment, a tissue to be treated is an inflamedtissue and the angiogenesis to be inhibited is inflamed tissueangiogenesis where there is neovascularization of inflamed tissue. Inthis class the method contemplates inhibition of angiogenesis inarthritic tissues, such as in a patient with chronic articularrheumatism, in immune or non-immune inflamed tissues, in psoriatictissue and the like.

The patient treated in the present invention in its many embodiments isdesirably a human patient, although it is to be understood that theprinciples of the invention indicate that the invention is effectivewith respect to all vertebrate species, including mammals, which areintended to be included in the term “patient”. In this context, a mammalis understood to include any mammalian species in which treatment ofdiseases associated with angiogenesis is desirable, particularlyagricultural and domestic mammalian species.

The methods of the present invention are particularly useful in thetreatment of warm-blooded vertebrates. Therefore, the invention concernsmammals and birds.

More particularly, contemplated is the treatment of mammals such ashumans, as well as those mammals of importance due to being endangered(such as Siberian tigers), of economic importance (animals raised onfarms for consumption by humans) and/or social importance (animals keptas pets or in zoos) to humans, for instance, carnivores other thanhumans (such as cats and dogs), swine (pigs, hogs, and wild boars),ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison,and camels), and horses. Also contemplated is the treatment of birds,including the treatment of those kinds of birds that are endangered,kept in zoos, as well as fowl, and more particularly domesticated fowl,i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, andthe like, as they are also of economic importance to humans. Thus,contemplated is the treatment of livestock, including, but not limitedto, domesticated swine (pigs and hogs), ruminants, horses, poultry, andthe like.

In another related embodiment, a tissue to be treated is a retinaltissue of a patient with diabetic retinopathy, macular degeneration orneovascular glaucoma and the angiogenesis to be inhibited is retinaltissue angiogenesis where there is neovascularization of retinal tissue.

In an additional related embodiment, a tissue to be treated is a tumortissue of a patient with a solid tumor, a metastasis, a skin cancer, ahemangioma or angiofibroma and the like cancer, and the angiogenesis tobe inhibited is tumor tissue angiogenesis where there isneovascularization of a tumor tissue.

Inhibition of tumor tissue angiogenesis is a particularly preferredembodiment because of the important role neovascularization plays intumor growth. In the absence of neovascularization of tumor tissue, thetumor tissue does not obtain the required nutrients, slows in growth,ceases additional growth, regresses and ultimately becomes necroticresulting in killing of the tumor. Stated differently, the presentinvention provides for a method of modulating tumor neovascularizationby modulating tumor angiogenesis according to the present methods.Similarly, the invention provides a method of modulating tumor growth bypracticing the angiogenesis-modulating methods.

The methods are also particularly effective against the formation ofmetastases because (1) their formation requires vascularization of aprimary tumor so that the metastatic cancer cells can exit the primarytumor and (2) their establishment in a secondary site requiresneovascularization to support growth of the metastases.

In a related embodiment, the invention pertains to the practice of themethod in conjunction with other therapies such as conventionalchemotherapy or surgery directed against solid tumors and for control ofestablishment of metastases. The administration of angiogenesisinhibitor can be conducted before, during or after chemotherapy orsurgery. For example, the angiogenesis inhibition methods of the presentinvention can be practiced for chronic maintenance. As additionalexample, the angiogenesis inhibition methods of the present inventioncan be practiced after a regimen of chemotherapy at times where thetumor tissue will be responding to the toxic assault by inducingangiogenesis to recover by the provision of a blood supply and nutrientsto the tumor tissue. As a further example, the angiogenesis inhibitionmethods of the present invention can be practiced after surgery wheresolid tumors have been removed as a prophylaxis against metastases.

The present method for modulating angiogenesis in a tissue contemplatescontacting a tissue in which angiogenesis is occurring, or is at riskfor occurring, with a composition comprising a therapeutically effectiveamount of an ECRTP/DEP-1 modulator capable of binding the ECRTP/DEP-1.Thus, the method comprises administering to a patient a therapeuticallyeffective amount of a physiologically tolerable composition containingan ECRTP/DEP-1 modulator of the invention.

The dosage ranges for the administration of the ECRTP/DEP-1 modulatordepend upon the form of the modulator, and its potency, as describedfurther herein, and are amounts large enough to produce the desiredeffect in which angiogenesis and the disease symptoms mediated byangiogenesis are ameliorated. The dosage should not be so large as tocause adverse side effects, such as hyperviscosity syndromes, pulmonaryedema, congestive heart failure, and the like. Generally, the dosagewill vary with the age, condition, sex and extent of the disease in thepatient and can be determined by one of skill in the art. The dosage canalso be adjusted by the individual physician in the event of anycomplication.

A therapeutically effective amount is an amount of an ECRTP/DEP-1receptor modulator sufficient to produce a measurable inhibition ofangiogenesis in the tissue being treated, i.e., anangiogenesis-modulating amount. Inhibition of angiogenesis can bemeasured in situ by immunohistochemistry, as described herein, or byother methods known to one skilled in the art.

Insofar as an ECRTP/DEP-1 modulator can take the form of an ECRTP/DEP-1ligand mimetic, and an anti-ECRTP/DEP-1 monoclonal antibody, or fragmentthereof, it is to be appreciated that the potency, and therefore anexpression of a “therapeutically effective” amount can vary. However, asshown by the present assay methods, one skilled in the art can readilyassess the potency of a candidate ECRTP/DEP-1 modulator of the presentinvention.

ECRTP/DEP-1 modulation can be measured by a variety of means includinginhibition of angiogenesis in the mouse corneal assay for angiogenesisdescribed herein, binding of natural ligand or monoclonal antibody to anECRTP/DEP-1 as described herein, and the like assays.

A preferred ECRTP/DEP-1 modulator has the ability to substantially bindto an ECRTP/DEP-1 in solution at modulator concentrations of less thanone (1) micro molar (μM), preferably less than 0.1 μM, and morepreferably less than 0.01 μM. By “substantially” is meant that at leasta 50 percent reduction in endothelial cell proliferation and migrationis observed by modulation in the presence of an ECRTP/DEP-1 modulator,and at 50% reduction is referred to herein as an IC50 value.

A therapeutically effective amount of an ECRTP/DEP-1 modulator of thepresent invention in the form of a monoclonal antibody, or fragmentthereof, is typically an amount such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.01 microgram (ug) per milliliter (mL) toabout 100 ug/mL, preferably from about 1 ug/mL to about 5 ug/mL, andusually about 5 ug/mL. For example, for Mab ECRTP/DEP-1 (MW=about 150kDa), 10 μg/mL≈67×10⁻⁹ M. Stated differently, the dosage can vary fromabout 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg toabout 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg,in one or more dose administrations daily, for one or several days.

A therapeutically effective amount of an ECRTP/DEP-1 modulator of thepresent invention in the form of a polypeptide is typically an amount ofpolypeptide such that when administered in a physiologically tolerablecomposition is sufficient to achieve a plasma concentration of fromabout 0.001 microgram (μg) per milliliter (mL) to about 10 μg/mL,preferably from about 0.05 μg/mL to about 1.0 ug/mL. Based on apolypeptide having a mass of about 15,000 grams per mole (i.e. 15,000Da), the preferred plasma concentration in molarity is from about 0.0001micro molar (μM) to about 1 milli molar (mM). Stated differently, thedosage per body weight can vary from about 0.01 mg/kg to about 30 mg/kg,and preferably from about 0.05 mg/kg to about 20 mg/kg, in one or moredose administrations daily, for one or several days.

The monoclonal antibodies or polypeptides of the invention can beadministered parenterally by injection or by gradual infusion over time.Although the tissue to be treated can typically be accessed in the bodyby systemic administration and therefore most often treated byintravenous administration of therapeutic compositions, other tissuesand delivery means are contemplated where there is a likelihood that thetissue targeted contains the target molecule. Thus, monoclonalantibodies or polypeptides of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intra-cavity, transdermally, and can be delivered by peristaltic means.

The therapeutic compositions containing a monoclonal antibody or apolypeptide of the present invention are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgement of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

C. Therapeutic Compositions

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with an ECRTP/DEP-1 modulator as describedherein, dissolved or dispersed therein as an active ingredient. In apreferred embodiment, the therapeutic ECRTP/DEP-1 modulator compositionis not immunogenic when administered to a mammal or human patient fortherapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions; however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

A therapeutic composition contains an angiogenesis-modulating amount ofan ECRTP/DEP-1 modulator of the present invention, typically formulatedto contain an amount of at least 0.1 weight percent of modulator perweight of total therapeutic composition. A weight percent is a ratio byweight of modulator to total composition. Thus, for example, 0.1 weightpercent is 0.1 grams of inhibitor per 100 grams of total composition.

D. Modulators of ECRTP/DEP-1

ECRTP/DEP-1 modulators are used in the present methods for modulatingECRTP/DEP-1 activity in tissues, including modulating angiogenesis intissues. Thus, as used herein, the terms “modulate”, “modulating”, and“modulator” are meant to be construed to encompass inhibiting, blocking,promoting, stimulating, agonising, antagonizing, or otherwise affectingECRTP/DEP-1 activity in tissues.

Such modulators can take a variety of forms that include compounds whichinteract with the ECRTP/DEP-1 in a manner such that functionalinteractions with natural ECRTP/DEP-1 ligands are mimicked, stimulatedand/or inhibited, such as, for example, dimerization of ECRTP/DEP-1.Exemplary modulators include analogs of an ECRTP/DEP-1 natural ligandbinding site on an ECRTP/DEP-1, mimetics of a natural ligand of anECRTP/DEP-1 that mimic the structural region involved in anECRTP/DEP-1-receptor ligand binding interactions, polypeptides having asequence corresponding to the domain of a natural ligand of anECRTP/DEP-1, and antibodies which immunoreact with either an ECRTP/DEP-1or the natural ligand, all of which exhibit modulator activity asdefined herein.

1. Polypeptides

In one embodiment, the invention contemplates ECRTP/DEP-1 modulators inthe form of polypeptides. A polypeptide (peptide) ECRTP/DEP-1 modulatorinteracts with the extracellular domain of ECRTP/DEP-1 and promotesdimerization of ECRTP/DEP-1. A preferred ECRTP/DEP-1 modulator peptidecorresponds in sequence to the natural ligand and promotes orantagonizes dimerization of ECRTP/DEP-1 ECRTP/DEP-1.

In one embodiment, a polypeptide of the present invention comprises nomore than about 100 amino acid residues, preferably no more than about60 residues, more preferably no more than about 30 residues. Peptidescan be linear or cyclic. Thus, it should be understood that a subjectpolypeptide need not be identical to the amino acid residue sequence ofan ECRTP/DEP-1 natural ligand, so long as it includes required bindingsequences and is able to function as an ECRTP/DEP-1 modulator in anassay such as is described herein.

A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide which is an ECRTP/DEP-1 modulator. Such apolypeptide can be subject to various changes, substitutions,insertions, and deletions where such changes provide for certainadvantages in its use. In this regard, an ECRTP/DEP-1 modulatorpolypeptide of the present invention corresponds to, rather than isidentical to, the sequence of the natural ligand where one or morechanges are made and it retains the ability to function as anECRTP/DEP-1 modulator in one or more of the assays as defined herein.Thus, a polypeptide can be in any of a variety of forms of peptidederivatives, that include amides, conjugates with proteins, cyclizedpeptides, polymerized peptides, analogs, fragments, chemically modifiedpeptides, and the like derivatives.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to a sequence of the natural ligand ofthe ECRTP/DEP-1 in which one or more residues have been conservativelysubstituted with a functionally similar residue and which displays theECRTP/DEP-1 modulator activity as described herein. Examples ofconservative substitutions include the substitution of one non-polar(hydrophobic) residue such as isoleucine, valine, leucine or methioninefor another; the substitution of one polar (hydrophilic) residue foranother such as between arginine and lysine, between glutamine andasparagine, between glycine and serine; the substitution of one basicresidue such as lysine, arginine or histidine for another; or thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite inhibitionactivity.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups canbe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups can be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine canbe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline can be substituted for proline;5-hydroxylysine can be substituted for lysine; 3-methylhistidine can besubstituted for histidine; homoserine can be substituted for serine; andornithine can be substituted for lysine. Polypeptides of the presentinvention also include any polypeptide having one or more additionsand/or deletions or residues relative to the sequence of a polypeptidewhose sequence is shown herein, so long as the requisite activity ismaintained. The term “fragment” refers to any subject polypeptide havingan amino acid residue sequence shorter than that of a polypeptidedisclosed herein.

When a polypeptide of the present invention has a sequence that is notidentical to the sequence of an ECRTP/DEP-1 natural ligand, it istypically because one or more conservative or non-conservativesubstitutions have been made, usually no more than about 30 numberpercent, and preferably no more than 10 number percent of the amino acidresidues are substituted. Additional residues can also be added ateither terminus of a polypeptide for the purpose of providing a “linker”by which the polypeptides of the present invention can be convenientlyaffixed to a label or solid matrix, or carrier. Labels, solid matricesand carriers that can be used with the polypeptides of the presentinvention are described hereinbelow.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues, but do not formECRTP/DEP-1 ligand epitopes. Typical amino acid residues used forlinking are tyrosine, cysteine, lysine, glutamic and aspartic acid, orthe like. In addition, a subject polypeptide can differ, unlessotherwise specified, from the natural sequence of an ECRTP/DEP-1 ligandby the sequence being modified by terminal-NH2 acylation, e.g.,acetylation, or thioglycolic acid amidation, byterminal-carboxylamidation, e.g., with ammonia, methylamine, and thelike terminal modifications. Terminal modifications are useful, as iswell known, to reduce susceptibility by proteinase digestion, andtherefore serve to prolong half life of the polypeptides in solutions,particularly biological fluids where proteases can be present. In thisregard, polypeptide cyclization is also a useful terminal modification,and is particularly preferred also because of the stable structuresformed by cyclization and in view of the biological activities observedfor such cyclic peptides as described herein.

Any peptide of the present invention can be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable ofthe peptides with the peptides of the present invention includeinorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid(HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid or the like. HCl and TFA salts areparticularly preferred.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-di- and tri-alkyl and aryl amines (e.g. triethylamine, diisopropylamine, methyl amine, dimethyl amine and the like), and optionallysubstituted ethanolamines (e.g. ethanolamine, diethanolamine and thelike).

A peptide of the present invention, also referred to herein as a subjectpolypeptide, can be synthesized by any of the techniques that are knownto those skilled in the polypeptide art, including recombinant DNAtechniques. Synthetic chemistry techniques, such as a solid-phaseMerrifield-type synthesis, are preferred for reasons of purity,antigenic specificity, freedom from undesired side products, ease ofproduction and the like. An excellent summary of the many techniquesavailable can be found in Steward et al., “Solid Phase PeptideSynthesis”, W. H. Freeman Co., San Francisco, 1969; Bodanszky, et al.,“Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976; J.Meienhofer, “Hormonal Proteins and Peptides”, Vol. 2, p. 46, AcademicPress (New York), 1983; Merrifield, Adv Enzymol, 32:221-96,1969; Fieldset al., Int. J. Peptide Protein Res., 35:161-214, 1990; and U.S. Pat.No. 4,244,946 for solid phase peptide synthesis, and Schroder et al.,“The Peptides”, Vol. 1, Academic Press (New York), 1965 for classicalsolution synthesis, each of which is incorporated herein by reference.Appropriate protective groups usable in such synthesis are described inthe above texts and in J. F. W. McOmie, “Protective Groups in OrganicChemistry”, Plenum Press, New York, 1973, which is incorporated hereinby reference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

Using a solid phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the final linearpolypeptide.

The resultant linear polypeptides prepared for example as describedabove can be reacted to form their corresponding cyclic peptides. Anexemplary method for cyclizing peptides is described by Zimmer et al.,Peptides 1992, pp. 393-394, ESCOM Science Publishers, B. V., 1993.Typically, tertbutoxycarbonyl protected peptide methyl ester isdissolved in methanol and sodium hydroxide solution are added and theadmixture is reacted at 20° C. to hydrolytically remove the methyl esterprotecting group. After evaporating the solvent, the tertbutoxycarbonylprotected peptide is extracted with ethyl acetate from acidified aqueoussolvent. The tertbutoxycarbonyl protecting group is then removed undermildly acidic conditions in dioxane cosolvent. The unprotected linearpeptide with free amino and carboxy termini so obtained is converted toits corresponding cyclic peptide by reacting a dilute solution of thelinear peptide, in a mixture of dichloromethane and dimethylformamide,with dicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazoleand N-methylmorpholine. The resultant cyclic peptide is then purified bychromatography.

2. Antibodies

The present invention describes, in one embodiment, ECRTP/DEP-1modulators in the form of antibodies, including monoclonal antibodies,which immunoreact with an ECRTP/DEP-1 and bind the ECRTP/DEP-1 tomodulate receptor activity as described herein. The invention alsodescribes cell lines which produce the antibodies, methods for producingthe cell lines, and methods for producing the antibodies, includingmonoclonal antibodies.

An antibody of the present invention can comprise an antibody moleculethat 1) immunoreact with isolated ECRTP/DEP-1, and 2) bind to theECRTP/DEP-1 to modulate its biological function. Preferably, an antibodyof the present invention preferentially binds the ECRTP/DEP-1ectodomain, which comprises amino acids 1-351 of ECRTP/DEP-1. Morepreferably, an antibody of the present invention preferentially binds aneight amino acid epitope having the sequence n-QSRDTEVL-c (SEQ ID NO:1), or an eight amino acid epitope having an analog sequence of thesequence n-QSRDTEVL-c (SEQ ID NO: 1), the term “analog” as definedherein, of the ECRTP/DEP-1 ectodomain.

Preferred monoclonal antibodies which preferentially bind to ECRTP/DEP-1include a monoclonal antibody having the immunoreaction characteristicsof Mab ECRTPAb-1, having molecular weight of about 150 kDa respectivelyand which binds to the ectodomain of the ECRTP/DEP-1, as is describedherein below. Mab ECRTPAb-1 is preferably secreted by hybridoma cellline ATCC HB12570. The hybridoma cell line ATCC HB12570 was depositedpursuant to Budapest Treaty requirements with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va.,20110-2209, U.S.A., on Sep. 18, 1998.

The term “antibody or antibody molecule” in the various grammaticalforms is used herein as a collective noun that refers to a population ofimmunoglobulin molecules and/or immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antibodycombining site or paratope. An “antibody combining site” is thatstructural portion of an antibody molecule comprised of heavy and lightchain variable and hypervariable regions that specifically bindsantigen.

Exemplary antibodies for use in the present invention are intactimmunoglobulin molecules, substantially intact immunoglobulin molecules,single chain immunoglobulins or antibodies, those portions of animmunoglobulin molecule that contain the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), and alsoreferred to as antibody fragments.

Indeed, as described in the Examples set forth below, an Fab fragment,that is, a monovalent fragment, of the Mab ECRTPAb-1 releases densityarrest. Thus, it is contemplated to be within the scope of the presentinvention that such a monovalent modulator is used to promoteangiogenesis, or to promote endothelial cell migration andproliferation, or to release inhibitory influences on endothelial cellsto serve as an adjunctive to other angiogenic stimuli. Thus, the terms“modulate”, “modulating”, and “modulator” are meant to be construed toencompass such promotion.

The phrase “monoclonal antibody” in its various grammatical forms refersto a population of antibody molecules that contain only one species ofantibody combining site capable of immunoreacting with a particularepitope. A monoclonal antibody thus typically displays a single bindingaffinity for any epitope with which it immunoreacts. A monoclonalantibody can therefore contain an antibody molecule having a pluralityof antibody combining sites, each immunospecific for a differentepitope, e.g., a bispecific monoclonal antibody.

A monoclonal antibody is typically composed of antibodies produced byclones of a single cell called a hybridoma that secretes (produces) onlyone kind of antibody molecule. The hybridoma cell is formed by fusing anantibody-producing cell and a myeloma or other self-perpetuating cellline. The preparation of such antibodies was first described by Kohlerand Milstein, Nature 256:495-497 (1975), which description isincorporated by reference. Additional methods are described by Zola,Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987).The hybridoma supernates so prepared can be screened for the presence ofantibody molecules that immunoreact with an ECRTP/DEP-1 and forinhibition of an ECRTP/DEP-1 to activate its biological function.

Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell lineis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a source of an ECRTP/DEP-1, as described by Chereshet al., J. Biol Chem, 262:17703-17711 (1987).

It is preferred that the myeloma cell line used to prepare a hybridomabe from the same species as the lymphocytes. Typically, a mouse of thestrain 129 GIX+ is the preferred mammal. Suitable mouse myelomas for usein the present invention include thehypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3×63-Ag8.653, and Sp2/0-Ag14 that are available from the ATCC,Manassas, Va., under the designations CRL 1580 and CRL 1581,respectively.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 1500. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody of the present inventionare identified using the enzyme linked immunosorbent assay (ELISA)described in the Examples.

A monoclonal antibody of the present invention can also be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate specificity. The culture is maintained under conditions andfor a time period sufficient for the hybridoma to secrete the antibodymolecules into the medium. The antibody-containing medium is thencollected. The antibody molecules can then be further isolated by wellknown techniques. Media useful for the preparation of these compositionsare both well known in the art and commercially available and includesynthetic culture media, inbred mice and the like. An exemplarysynthetic medium is Dulbecco's minimal essential medium (DMEM—Dulbeccoet al., Virol 8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mMglutamine, and 20% fetal calf serum. An exemplary inbred mouse strain isthe Balb/C.

Other methods of producing a monoclonal antibody, a hybridoma cell, or ahybridoma cell culture are also well known. See, for example, the methodof isolating monoclonal antibodies from an immunological repertoire asdescribed by Sastry, et al., Proc Natl Acad Sci USA 86:5728-5732 (1989);and Huse et al., Science 246:1275-1281 (1989).

Also contemplated by the present invention is the hybridoma cell, andcultures containing a hybridoma cell that produce a monoclonal antibodyof the present invention. Particularly preferred is the hybridoma cellline that secretes monoclonal antibody Mab ECRTPAb-1 as described in theExamples presented below and as designated ATCC HB12570. Mab ECRTPAb-1was prepared as described in the Examples. The invention thuscontemplates, in one embodiment, a monoclonal antibody that has theimmunoreaction characteristics of Mab ECRTPAb-1.

It is also possible to determine, without undue experimentation, if amonoclonal antibody has the same (i.e., equivalent) specificity(immunoreaction characteristics) as a monoclonal antibody of the presentinvention by ascertaining whether the former prevents the latter frombinding to a preselected target molecule. If the monoclonal antibodybeing tested competes with the monoclonal antibody of the invention, asshown by a decrease in binding by the monoclonal antibody of theinvention in standard competition assays for binding to the targetmolecule when present in the solid phase, then it is likely that the twomonoclonal antibodies bind to the same, or a closely related, epitope. Apreferred target molecule comprises a polypeptide fragment of theECRTP/DEP-1 ectodomain includes an eight amino acid epitope having thesequence n-QSRDTEVL-c (SEQ ID NO: 1), or an eight amino acid epitopehaving an analog sequence of the sequence n-QSRDTEVL-c (SEQ ID NO: 1),the term “analog” as defined herein.

Still another way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to pre-incubatethe monoclonal antibody of the invention with the target molecule withwhich it is normally reactive, and then add the monoclonal antibodybeing tested to determine if the monoclonal antibody being tested isinhibited in its ability to bind the target molecule. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody of the invention. A preferred target molecule comprises apolypeptide fragment of the ECRTP/DEP-1 ectodomain includes an eightamino acid epitope having the sequence n-QSRDTEVL-c (SEQ ID NO: 1), oran eight amino acid epitope having an analog sequence of the sequencen-QSRDTEVL-c (SEQ ID NO: 1), the term “analog” as defined herein.

An additional way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to determinethe amino acid residue sequence of the CDR regions of the antibodies inquestion. Antibody molecules having identical, or functionallyequivalent, amino acid residue sequences in their CDR regions have thesame binding specificity. Methods for sequencing polypeptides are wellknown in the art.

The immunospecificity of an antibody, its target molecule bindingcapacity, and the attendant affinity the antibody exhibits for theepitope, are defined by the epitope with which the antibodyimmunoreacts. The epitope specificity is defined at least in part by theamino acid residue sequence of the variable region of the heavy chain ofthe immunoglobulin that comprises the antibody, and in part by the lightchain variable region amino acid residue sequence. Use of the terms“having the binding specificity of” or “having the binding preferenceof” indicates that equivalent monoclonal antibodies exhibit the same orsimilar immunoreaction (binding) characteristics and compete for bindingto a preselected target molecule. Preferably, an antibody of the presentinvention preferentially binds an eight amino acid epitope having thesequence n-QSRDTEVL-c (SEQ ID NO: 1), or an eight amino acid epitopehaving an analog sequence of the sequence n-QSRDTEVL-c (SEQ ID NO: 1),the term “analog” as defined herein, of the ECRTP/DEP-1 ectodomain.

Humanized monoclonal antibodies offer particular advantages over murinemonoclonal antibodies, particularly insofar as they can be usedtherapeutically in humans. Specifically, human antibodies are notcleared from the circulation as rapidly as “foreign” antigens, and donot activate the immune system in the same manner as foreign antigensand foreign antibodies. Methods of preparing “humanized” antibodies aregenerally well known in the art, and can readily be applied to theantibodies of the present invention. Thus, the invention contemplates,in one embodiment, a monoclonal antibody of the present invention thatis humanized by grafting to introduce components of the human immunesystem without substantially interfering with the ability of theantibody to bind antigen. Humanized antibodies can also be producedusing animals engineering to produce humanized antibodies, such as thoseavailable from Medarex of Annandale, N.J. (mice) and Abgenix, Inc., ofFremont, Calif. (mice).

The use of a molecular cloning approach to generate antibodies,particularly monoclonal antibodies, and more particularly single chainmonoclonal antibodies, is also contemplated. The production of singlechain antibodies has been described in the art, see e.g., U.S. Pat. No.5,260,203, the contents of which are herein incorporated by reference.For this, combinatorial immunoglobulin phagemid or phage-displayedlibraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning on endothelial tissue. This approach can also beused to prepare humanized antibodies. The advantages of this approachover conventional hybridoma techniques are that approximately 10⁴ timesas many antibodies can be produced and screened in a single round, andthat new specificities are generated by H and L chain combination in asingle chain, which further increases the chance of finding appropriateantibodies. Thus, an antibody of the present invention, or a“derivative” of an antibody of the present invention pertains to asingle polypeptide chain binding molecule which has binding specificityand affinity substantially similar to the binding specificity andaffinity of the light and heavy chain aggregate variable region of anantibody described herein, such as ECRTP/DEP-1. Preferably, an antibodyof the present invention preferentially binds an eight amino acidepitope having the sequence n-QSRDTEVL-c (SEQ ID NO: 1), or an eightamino acid epitope having an analog sequence of the sequencen-QSRDTEVL-c (SEQ ID NO: 1), the term “analog” as defined herein, of theECRTP/DEP-1 ectodomain.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species(scFv), one heavy- and one light-chain variable domain can be covalentlylinked by a flexible peptide linker such that the light and heavy chainscan associate in a “dimeric” structure analogous to that in a two-chainFv species. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

Using a phage-displayed approach for the production of antibodies, scFvantibody clones that bind to the ECRTP/DEP-1 ectodomain have beenidentified. This was accomplished by competing off those phage thatdisplayed antibodies using ECRTPAb-1 disclosed herein. Fv regions aresequenced a bivalent functional reagents are designed and tested in ascreening assay of the present invention disclosed herein below. Thus, apreferred source for an antibody, or derivative or fragment thereof, isa recombinant phage-displayed antibody library. The recombinant phagecan comprise antibody encoding nucleic acids isolated from any suitablevertebrate species, including mammalian species such as mouse and rat;but preferably comprises antibody encoding nucleic acids isolated fromhuman. Such antibodies are thus already “humanized”.

3. Other Modulators

It is also envisioned that previously described and newly discoveredangiogenesis inhibiting or endothelial cell growth suppressing chemicalcompounds are modulators of ECRTP/DEP-1 activity in tissues. Examples ofsuch compounds include, but are not limited to, angiostatin, endostatinand thrombospondin. Accordingly, such compounds can be used in themodulation of ECRTP/DEP-1 activity in tissues, according to the methodsof the present invention.

Given the disclosure of the ECRTP/DEP-1 activity in tissues herein, itis also contemplated that as yet undefined chemical compounds can beused to modulate ECRTP/DEP-1 activity in tissues in accordance with themethods of the present invention. In one embodiment, a modulator of thepresent invention interacts with an eight amino acid epitope having thesequence n-QSRDTEVL-c (SEQ ID NO: 1) of the ECRTP/DEP-1 ectodomain. Inanother embodiment, a modulator of the present invention interacts withan intracellular catalytic domain of ECRTP/DEP-1. The identification ofsuch modulators is provided through the description of screening assaysdirected to ECRTP/DEP-1 activity in tissues presented herein.

D. Screening Assay

Skilled artisans will understand that the disclosure herein of thelocalization and function of the ECRTP/DEP-1, and in vitro assaysrelating to such localization and function, provides opportunities toscreen for compounds that modulate, whether partially or completely, thefunctional activity of the ECRTP/DEP-1. In this context, “modulate” isintended to mean that the subject compound increases or decreases one ormore functional activities of the ECRTP/DEP-1, such as but not limitedto ECRTP/DEP-1 activity in cell growth, cell survival and angiogenesis.

Further, the screening assays illustrated in the Examples below includebiochemical assays (e.g., measuring effects of anti-ECRTP/DEP-1monoclonal antibodies on ECRTP/DEP-1 activity), and cellular in vitroassays (e.g., measuring the effects of ECRTP/DEP-1 over-expression onendothelial cell proliferation and migration and/or evaluatingECRTP/DEP-1 phosphorylation). The illustrative biochemical assays can beparticularly useful in screening for compounds modulating an ECRTP/DEP-1activity, while the cellular assays can be particularly useful inscreening for compounds completely altering an ECRTP/DEP-1 activity.Thus, until the disclosure herein of the role of the ECRTP/DEP-1 inregulating cell growth, cell survival, endothelial cell proliferationand migration and in regulating angiogenesis, a motivation to screen forcompounds that modulate ECRTP/DEP-1 activity was lacking in the priorart.

Those skilled in the art will understand that binding of a ligand at amolecular binding site can be modulated in a direct matter (e.g., byblocking the site), as well as modulated in an indirect manner (e.g., byconformational changes induced following binding of a second, i.e.,different, ligand at a distant site). In this regard, it is likely thatthe binding site specificity of an ECRTP/DEP-1 for its endogenous ligandcan be completely modulated or altered (i.e., to bind a differentligand) by agents that bind at distant sites in the ECRTP/DEP-1.Examples of compounds that can be screened in the latter several assaysinclude at least nucleic acids (e.g., DNA oligonucleotide aptamers thatbind proteins and alter their functions), proteins, antibodies andantibody fragments, carbohydrates, lectins, organic chemicals, and thelike. Such screening assays can be useful for identifying candidatetherapeutic agents that can provide drugs useful in animals and humans.

It is still further understood that due to the significance of theECRTP/DEP-1 in cell growth, cell survival, endothelial cell migrationand proliferation, in density induced growth arrest, and in modulationof angiogenesis, innate regulatory mechanisms exist in cells forregulating their activity by binding to an ECRTP/DEP-1, or to complexescontaining an ECRTP/DEP-1. Such regulatory factors can include, atleast: (a) cofactors that bind to the complex and exert regulatoryaction by destabilizing or stabilizing the complex; (b) agents thatmodulate or alter the activity of the complex by inducing conformationalchanges in the ECRTP/DEP-1 as they are bound in a complex; (c) enzymesthat inactivate one or both members of a complex; and (d) cellularcontrol factors (e.g., signal transduction second messengers,transcription regulating factors, DNA replication factors and the like)that bind an ECRTP/DEP-1 or ECRTP/DEP-1 complexes and modulate or alterfunctional activity. Those skilled in the art will recognize that thefunctional regions of an ECRTP/DEP-1 represent particularly attractivetargets for three-dimensional molecular modeling and for construction ofmimetic compounds, e.g., organic chemicals constructed to mimic thethree-dimensional interactions between the ECRTP/DEP-1 and itsendogenous binding partner, or other binding partner.

Utilizing the methods and compositions of the present invention,screening assays for the testing of candidate substances can be derived.A candidate substance is a substance which potentially can modulateendothelial cell growth, cell survival, cell migration andproliferation, density induced growth arrest and/or angiogenesis, and/orECRTP/DEP-1 phosphorylation, by binding or other intramolecularinteraction, with an ECRTP/DEP-1 that modulates endothelial cell growth,cell survival, cell migration and proliferation, density induced growtharrest and angiogenesis.

Thus, a method of screening candidate substances for an ability tomodulate ECRTP/DEP-1 biological activity is also disclosed. The methodcomprises establishing a test sample comprising an ECRTP/DEP-1polypeptide or fragment thereof; administering a candidate substance tothe test sample; and measuring the interaction, effect, or combinationthereof, of the candidate substance on the test sample to therebydetermine the ability of the candidate substance to modulate ECRTP/DEP-1biological activity.

The present invention also provides a process of screening substancesfor their ability to modulate or alter cell growth, cell survival,endothelial cell migration and proliferation, density induced growtharrest and/or angiogenesis and/or ECRTP/DEP-1 phosphorylation comprisingthe steps of providing a cell that contains a functional ECRTP/DEP-1 andtesting the ability of selected substances to modulate or alter cellgrowth, cell survival, migration or proliferation of that cell, densityinduced growth arrest of the cell, or initiation of angiogenesis in thecell and/or evaluating ECRTP/DEP-1 phosphorylation in the cell.

A screening assay of the present invention generally involvesdetermining the ability of a candidate substance to affect cell growth,cell survival, endothelial cell migration and proliferation, densityinduced growth arrest and/or angiogenesis, and/or ECRTP/DEP-1phosphorylation in a target cell, such as the screening of candidatesubstances to identify those that modulate or alter cell growth, cellsurvival, endothelial cell migration and proliferation, density inducedgrowth arrest and/or angiogenesis, and/or ECRTP/DEP-1 phosphorylation.Target cells can be either naturally occurring cells known to contain anECRTP/DEP-1 or transfected cell produced in accordance with a process oftransfection set forth herein and as are known in the art.

Thus, in one embodiment a method of screening a candidate substance foran ability to modulate a receptor tyrosine phosphatase in accordancewith the present invention comprises establishing test samplescomprising a receptor tyrosine phosphatase polypeptide; andadministering a candidate substance to the test samples; measuring theinteraction, effect, or combination thereof, of the candidate substanceon the test sample to thereby determine the ability of the candidatesubstance to modulate receptor tyrosine phosphatase biological activity.A preferred receptor tyrosine phosphatase comprises ECRTP/DEP-1.

In another embodiment, a method of screening a candidate substance foran ability to modulate a receptor tyrosine phosphatase in accordancewith the present invention comprises establishing a test samplecomprising a receptor tyrosine phosphatase; administering a candidatesubstance to the test sample; and measuring a receptor tyrosinephosphatase biological activity in the test sample; detectingphosphotyrosine residues on the receptor tyrosine phosphatase; anddetermining that the candidate substance modulates the receptor tyrosinephosphatase if the receptor tyrosine phosphatase biological activitymeasured for the test sample is greater or less than the receptortyrosine phosphatase biological activity measured for a control sampleand if the amount of phosphotyrosine residues on the receptor tyrosinephosphatase is greater or less than an amount of phosphotyrosineresidues on a receptor tyrosine phosphate derived from a control sample.A preferred receptor tyrosine phosphatase comprises ECRTP/DEP-1.

In yet another embodiment, a method of screening a candidate substancefor an ability to modulate a receptor tyrosine phosphatase in accordancewith the present invention comprises establishing replicate test andcontrol cultures of cells that express the ECRTP/DEP-1; administering acandidate compound to the cells in the test culture but not the controlculture; measuring ECRTP/DEP-1 activity in cells in the test and thecontrol cultures; and determining that the candidate compound modulatesthe ECRTP/DEP-1 activity in a cell if the ECRTP/DEP-1 activity measuredfor the test culture is greater or less than the ECRTP/DEP-1 activitymeasured for the control culture.

The screening assay methods of the present invention also pertain tocomparing the effect of a candidate compound to inhibit growth of cellsexpressing exogenous ECRTP/DEP-1 compared with those not expressingECRTP/DEP-1 and determining that the effect of altering ECRTP/DEP-1activity is responsible by demonstrating the lack of activity of thecandidate compound on cells not expressing ECRTP/DEP-1. The screeningassays of the present invention show changes in growth that areresponsive to changes in ECRTP/DEP-1 activity. For example, thescreening methods of the present invention are used to screen forbiologically active counter-receptors (ligands) by screening for growthinhibitory activity in biological fractions (plasma, cell lysates, cellmembrane extracted proteins) by comparing growth inhibition onCHO/ECRTP/DEP-1 to CHO parent cell lines.

In a cell-free system, a method of screening a candidate substance foran ability to modulate a receptor tyrosine phosphatase in accordancewith the present invention comprises establishing a control systemcomprising an ECRTP/DEP-1, or fragment thereof and a ligand wherein theECRTP/DEP-1 is capable of binding to the ligand; establishing a testsystem comprising the ECRTP/DEP-1, the ligand, and a candidate compound;measuring the binding affinity of the ECRTP/DEP-1 and the ligand in thecontrol and the test systems; and determining that the candidatecompound modulates ECRTP/DEP-1 activity in a cell-free system if thebinding affinity measured for the test system is less than or greaterthan the binding affinity measured for the control system. Optionally,the ligand comprises an antibody which preferentially binds theECRTP/DEP-1. In this case, it is preferred that the ligand comprise amonoclonal antibody.

In accordance with the present invention, a method of affinity screeningof candidate modulator substances, including but not limited toantibodies, is provided. The method comprises: (a) contacting acandidate modulator substance with an ECRTP/DEP-1 polypeptide orfragment thereof under conditions favorable to binding the candidatemodulator substance with an ECRTP/DEP-1 polypeptide or fragment thereofto form a complex therebetween; and (b) detecting the complex.

The complex can be detected in any suitable manner. For example, thecomplex can be detected via a label conjugated to the ECRTP/DEP-1polypeptide or fragment thereof; via a labeled reagent that specificallybinds to the complex subsequent to its formation; or via a competitionassay with a substance. The ECRTP/DEP-1 polypeptide fragment can be anECRTP/DEP-1 ectodomain fragment. Preferably, the ECRTP/DEP-1 ectodomainfragment comprises an eight amino acid epitope having the sequencen-QSRDTEVL-c (SEQ ID NO: 1), or an eight amino acid epitope having ananalog sequence of the sequence n-QSRDTEVL-c (SEQ ID NO: 1), the term“analog” as defined herein. Optionally, the ECRTP/DEP-1 polypeptide orfragment thereof is conjugated with a detectable label. In this case,the detecting step comprises: (i) separating the complex from unboundlabeled binding substance; and (ii) detecting the detectable label whichis present in the complex or which is unbound.

An antibody, or derivative or fragment thereof, can be screened as acandidate modulator substance. As noted above, a preferred source for anantibody, or derivative or fragment thereof, is a recombinantphage-displayed antibody library. The recombinant phage can compriseantibody encoding nucleic acids isolated from any suitable vertebratespecies, including mammalian species such as mouse and rat; butpreferably comprises antibody encoding nucleic acids isolated fromhuman. Such antibodies are thus already “humanized”.

In another aspect, the present invention pertains to a kit for use inthe aforementioned affinity screening method. The kit comprises abinding agent comprising a polypeptide fragment of the ECRTP/DEP-1ectodomain that comprises an eight amino acid epitope having thesequence n-QSRDTEVL-c (SEQ ID NO: 1), or an eight amino acid epitopehaving an analog sequence of the sequence n-QSRDTEVL-c (SEQ ID NO: 1),the term “analog” as defined herein, contained in a first container.Optionally, the binding agent can be immobilized to a solid phasesupport, or the kit can also comprise a solid phase support contained ina second container.

The kit can further comprise a reagent or indicator that comprises adetectable label, the indicator containing in another container.Alternatively, the binding agent can comprise a detectable label orindicator. Preferably, the indicator is a radioactive label or anenzyme, or other suitable indicator.

Another technique for drug screening which can be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT application WO84/03564, herein incorporated by reference. In this method, as appliedto the ECRTP/DEP-1 polypeptide, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with the ECRTP/DEP-1polypeptide, or fragments thereof, and washed. Bound ECRTP/DEP-1polypeptide is then detected by methods well known in the art. PurifiedECRTP/DEP-1 polypeptide can also be coated directly onto plates for usein the aforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

A screening assay of the present invention can also involve determiningthe ability of a candidate substance to modulate, i.e., inhibit orpromote ECRTP/DEP-1 biological activity and preferably, to therebymodulate the ECRTP/DEP-1 biological activity in target cells. Targetcells can be either naturally occurring cells known to contain apolypeptide of the present invention or transfected cells produced inaccordance with a process of transfection set forth herein above. Thetest samples can further comprise a cell or cell line that expresses theECRTP/DEP-1; the present invention also contemplates a recombinant cellline suitable for use in the exemplary method. Such cell lines can bemammalian, or human, or they can from another organism, including butnot limited to yeast. Exemplary assays including genetic screeningassays and molecular biology screens such as a yeast two-hybrid screenthat will effectively identify ECRTP/DEP-1-interacting genes importantfor endothelial cell migration and proliferation, density induced growtharrest, angiogenesis or other ECRTP/DEP-1-mediated cellular process. Oneversion of the yeast two-hybrid system has been described (Chien et al.,1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commerciallyavailable from Clontech (Palo Alto, Calif.).

A method of identifying modulators of the ECRTP/DEP-1 by rational drugdesign is also provided in accordance with the present invention. Themethod comprises the steps of designing a potential modulator for theECRTP/DEP-1 that will form non-covalent bonds with amino acids in thesubstrate binding site in the catalytic domain (an intracellular site)or with the ECRTP/DEP-1 ectodomain based upon the structure of theECRTP/DEP-1; synthesizing the modulator; and determining whether thepotential modulator modulates the activity of the ECRTP/DEP-1.

Thus, the present invention pertains to screening and rational drugdesign methods for both ectodomain interactions, as well as catalyticmodulators. For example, catalytic antagonists that release cells fromgrowth arrest through modulation of the catalytic function independentof the ectodomain can be screened and designed. Additionally, amodulator of the present invention can be screened for interaction withan eight amino acid epitope having the sequence n-QSRDTEVL-c (SEQ ID NO:1), or an eight amino acid epitope having an analog sequence of thesequence n-QSRDTEVL-c (SEQ ID NO: 1), the term “analog” as definedherein, of the ECRTP/DEP-1 ectodomain.

Modulators can be synthesized using techniques disclosed herein and asare known in the art. The determination of whether the modulatormodulates the biological activity of the ECRTP/DEP-1 is made inaccordance with the screening methods disclosed herein, or by otherscreening methods known in the art.

As is well known in the art, a screening assay provides a cell underconditions suitable for testing modulation or alteration of cell growth,cell endothelial cell migration and proliferation, density inducedgrowth arrest, angiogenesis, and/or ECRTP/DEP-1 phosphorylation. Theseconditions include but are not limited to pH, temperature, tonicity, thepresence of relevant factors involved in the cell cycle (e.g., growthfactors), and relevant modifications to the polypeptide such asglycosylation or prenylation. It is contemplated that an ECRTP/DEP-1 canbe expressed and utilized in a prokaryotic or eukaryotic cell. The hostcell can also be fractionated into sub-cellular fractions where thereceptor can be found. For example, cells expressing the polypeptide canbe fractionated into the nuclei, the endoplasmic reticulum, vesicles, orthe membrane surfaces of the cell.

pH is preferably from about a value of 6.0 to a value of about 8.0, morepreferably from about a value of about 6.8 to a value of about 7.8 and,most preferably about 7.4. In a preferred embodiment, temperature isfrom about 20° C. to about 50° C., more preferably from about 30° C. toabout 40° C. and, even more preferably about 37° C. Osmolality ispreferably from about 5 milliosmols per liter (mosm/L) to about 400mosm/l and, more preferably from about 200 milliosmols per liter toabout 400 mosm/l and, even more preferably from about 290 mosm/L toabout 310 mosm/L. The presence of factors can be required for the propertesting of endothelial cell migration and proliferation, density inducedgrowth arrest and/or angiogenesis in specific cells. Such factorsinclude, for example, the presence and absence (withdrawal) of growthfactor, interleukins, or colony stimulating factors.

E. Methods for Identifying Modulators of an ECRTP/DEP-1

The invention thus also pertains to assay methods for identifyingcandidate ECRTP/DEP-1 modulators. In these assay methods candidatemolecules are evaluated for their potency in agonising an ECRTP/DEP-1binding to natural ligands, and furthermore are evaluated for theirpotency in modulating angiogenesis in a tissue.

An exemplary assay measures angiogenesis in the chick chorioallantoicmembrane (CAM) and is referred to as the CAM assay. The CAM assay hasbeen described in detail by others, and further has been used to measureboth angiogenesis and neovascularization of tumor tissues. See Ausprunket al., Am J Pathol 79:597-618 (1975) and Ossonski et al., Cancer Res40:2300-2309 (1980).

The CAM assay is a well recognized assay model for in vivo angiogenesisbecause neovascularization of whole tissue is occurring, and actualchick embryo blood vessels are growing into the CAM or into the tissuegrown on the CAM. The CAM assay illustrates inhibition ofneovascularization based on both the amount and extent of new vesselgrowth. Furthermore, it is easy to monitor the growth of any tissuetransplanted upon the CAM, such as a tumor tissue. Finally, the assay isparticularly useful because there is an internal control for toxicity inthe assay system. The chick embryo is exposed to any test reagent, andtherefore the health of the embryo is an indication of toxicity.

F. Preparation of Targeting Agent/Toxin Compounds, IncludingImmunotoxins

Methods for the production of the target agent/toxin agent compounds ofthe invention are described herein. The targeting agents, such asantibodies, of the invention can be linked, or operatively attached, tothe toxins of the invention by either crosslinking or via recombinantDNA techniques, to produce, for example, targeted immunotoxins.

While the preparation of immunotoxins is, in general, well known in theart (see e.g., U.S. Pat. Nos. 4,340,535 and 5,776,427, and EP 44167,each of which incorporated herein by reference), certain advantages canbe achieved through the application of certain preferred technology,both in the preparation of the immunotoxins and in their purificationfor subsequent clinical administration. For example, while numeroustypes of disulfide-bond containing linkers are known which cansuccessfully be employed to conjugate the toxin moiety with thetargeting agent, certain linkers will generally be preferred over otherlinkers, based on differing pharmacologic characteristics andcapabilities. For example, linkers that contain a disulfide bond that issterically “hindered” are to be preferred, due to their greaterstability in vivo, thus preventing release of the toxin moiety prior tobinding at the site of action.

A wide variety of cytotoxic agents are known that can be conjugated toanti-endothelial cell antibodies. Examples include numerous usefulplant-, fungus- or even bacteria-derived toxins, which, by way ofexample, include various A chain toxins, particularly ricin A chain,ribosome inactivating proteins such as saporin or gelonin, α-sarcin,aspergillin, restrictocin, ribonucleases such as placental ribonuclease,angiogenic, diphtheria toxin, and pseudomonas exotoxin, to name just afew.

However, it can be desirable from a pharmacologic standpoint to employthe smallest molecule possible that nevertheless provides an appropriatebiological response. One can thus desire to employ smaller-chainpeptides which will provide an adequate anti-cellular response.

Alternatively, one can find that the application of recombinant DNAtechnology to the toxin moiety will provide additional significantbenefits in accordance the invention. For example, the cloning andexpression of biologically active toxin candidates has now beendescribed through the publications of others (O'Hare et al., FEBS Lett210:731 (1987); Lamb et al., Eur J Biochem 148:265-270 (1985); Hailinget al., Nucl Acids Res 13:8019-8033 (1985)), it is now possible toidentify and prepare smaller or otherwise variant peptides whichnevertheless exhibit an appropriate toxin activity. Moreover, the use ofcloned toxin candidates allows the application of site-directedmutagenesis, through which one can readily prepare and screen formutated peptides and obtain additional useful moieties for use inconnection with the present invention.

In cases where a releasable toxin is contemplated, one desires to have aconjugate that will remain intact under conditions found everywhere inthe body except the intended site of action, at which point it isdesirable that the conjugate have good “release” characteristics.Therefore, the particular crosslinking scheme, including the particularcrosslinking reagent used and the structures that are crosslinked, willbe of some significance.

Crosslinking reagents are used to form molecular bridges that tietogether functional groups of two different proteins (e.g., a toxin anda binding agent). To link two different proteins in a step-wise manner,heterobifunctional crosslinkers can be used which eliminate the unwantedhomopolymer formation. An exemplary heterobifunctional crosslinkercontains two reactive groups: one reacting with primary amine group(e.g., N-hydroxy succinimide) and the other reacting with a thiol group(e.g., pyridyl disulfide, maleimides, halogens, etc.). Through theprimary amine reactive group, the crosslinker can react with the lysineresidue(s) of one protein (e.g., the selected antibody or fragment) andthrough the thiol reactive group, the crosslinker, already tied up tothe first protein, reacts with the cysteine residue (free sulfhydrylgroup) of the other protein.

The spacer arm between these two reactive groups of any crosslinkers canhave various length and chemical composition. A longer spacer arm allowsa better flexibility of the conjugate components while some particularcomponents in the bridge (e.g., benzene group) can lend extra stabilityto the reactive group or an increased resistance of the chemical link tothe action of various aspects (e.g., disulfide bond resistant toreducing agents).

An exemplary crosslinking reagent is SMPT, which is a bifunctionalcrosslinker containing a disulfide bond that is “sterically hindered” byan adjacent benzene ring and methyl groups. It is believed that stearichindrance of the disulfide bond serves a function of protecting the bondfrom attack by thiolate anions such as glutathione which can be presentin tissues and blood, and thereby help in preventing decoupling of theconjugate prior to its delivery to the site of action by the bindingagent. The SMPT crosslinking reagent, as with many other knowncrosslinking reagents, lends the ability to crosslink functional groupssuch as the SH of cysteine or primary amines (e.g., the epsilon aminogroup of lysine). Another possible type of crosslinker includes theheterobifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidylgroup reacts with primary amino groups and the phenylazide (uponphotolysis) reacts non-selectively with any amino acid residue.

Although the “hindered” crosslinkers will generally be preferred in thepractice of the invention, non-hindered linkers can be employed andadvantages in accordance herewith nevertheless realized. Other usefulcrosslinkers, not considered to contain or generate a protecteddisulfide, include SATA, SPDP and 2-iminothiolane (Thorpe et al., CancerRes 47:5924-5931 (1987)). The use of such crosslinkers is wellunderstood in the art.

Once conjugated, it will be important to purify the conjugate so as toremove contaminants such as unconjugated toxin or targeting agent. It isimportant to remove unconjugated targeting agent to reduce undesiredtoxicity and to avoid the possibility of competition for the antigenbetween conjugated and unconjugated species. In general, the mostpreferred purification technique will incorporate the use of BlueSepharose with a gel filtration or gel permeation step. Blue Sepharoseis a column matrix composed of Cibacron Blue 3GA and agarose, which hasbeen found to be useful in the purification of immunoconjugates (Knowles& Thorpe, Anal. Biochem 120:440-443 (1987)). The use of Blue Sepharosecombines the properties of ion exchange with toxin binding to providegood separation of conjugated toxin from non-conjugated toxin. The BlueSepharose column allows the elimination of the free (non-conjugated)targeting agent (e.g., the antibody or fragment) from the conjugatepreparation. To eliminate the free (non-conjugated) toxin a molecularexclusion chromatography step is preferred using either conventional gelfiltration procedure or high performance liquid chromatography.

Standard recombinant DNA techniques that are well known to those ofskill in the art can be utilized to express nucleic acids encoding thetargeting agent/toxin compounds of the invention. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. DNA and RNA synthesiscan, additionally, be performed using an automated synthesizers (see,for example, the techniques described in Sambrook et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1989); and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, New York (1989).

When produced via recombinant DNA techniques such as those describedherein, the targeting agent/toxin compounds of the invention can bereferred to herein as “fusion proteins”. It is to be understood thatsuch fusion proteins contain at least a targeting agent and a toxicmoiety operatively attached, such that the fusion protein can be used inaccordance with the methods of the present invention. The fusionproteins can also include additional peptide sequences, such as peptidespacers which operatively attach the targeting agent and toxin compound,as long as such additional sequences do not appreciably affect thetargeting or toxin activities of the fusion protein.

Depending on the specific toxin compound used as part of the fusionprotein, it can be necessary to provide a peptide spacer operativelyattaching the targeting agent and the toxin compound which is capable offolding into a disulfide-bonded loop structure. Proteolytic cleavagewithin the loop would then yield a heterodimeric polypeptide wherein thetargeting agent and the toxin compound are linked by only a singledisulfide bond. See e.g., Lord et al., in Genetically Engineered Toxins(Ed. A. Frank, M. Dekker Publ., p. 183) (1992). An example of such atoxin is a Ricin A-chain toxin.

When certain other toxin compounds are utilized, a non-cleavable peptidespacer can be provided to operatively attach the targeting agent and thetoxin compound of the fusion protein. Toxins which can be used inconjunction with non-cleavable peptide spacers are those which can,themselves, be converted by proteolytic cleavage, into a cytotoxicdisulfide-bonded form (see e.g., Ogata et al., J Biol Chem256:20678-20685 (1990)). An example of such a toxin compound is aPseudomonas exotoxin compound.

Nucleic acids that can be utilized herein comprise nucleic acidsequences that encode a targeting agent of interest and nucleic acidsequences that encode a toxin agent of interest. Such targetagent-encoding and toxin agent-encoding nucleic acid sequences areattached in a manner such that translation of the nucleic acid yieldsthe targeting agent/toxin compounds of the invention.

Standard techniques, such as those described above can be used toconstruct expression vectors containing the above-described nucleicacids and appropriate transcriptional/translational control sequences. Avariety of host-expression vector systems can be utilized. These includebut are not limited to microorganisms such as bacteria (e.g., E. coli,B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNAor cosmid DNA expression vectors containing targeting agent/toxin codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing targeting agent/toxincoding sequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the targetingagent/toxin coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing the targetingagent/toxin coding sequences coding sequence; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter;lentiviral vectors).

In bacterial systems a number of expression vectors can beadvantageously selected depending upon the use intended for thetargeting agent/toxin compound being expressed. For example, when largequantities of targeting agent/toxin compound are to be produced for thegeneration of antibodies or to screen peptide libraries, vectors whichdirect the expression of high levels of fusion protein products that arereadily purified can be desirable. Such vectors include but are notlimited to the E. coli expression vector pUR278 (Ruther et al., EMBO J2:1791 (1983)), in which the targeting agent/toxin coding sequence canbe ligated individually into the vector in frame with the lac Z codingregion so that a fusion protein additionally containing a portion of thelac Z product is provided; pIN vectors (Inouye et al., Nucleic Acids Res13:3101-3109 (1985); Van Heeke et al., J Biol Chem 264:5503-5509(1989)); and the like. pGEX vectors can also be used to express foreignpolypeptides, such as the targeting agent/toxin compounds as fusionproteins additionally containing glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the targeting agent/toxin protein of the fusion protein can bereleased from the GST moiety.

In an insect system, Autograph californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The targeting agent/toxin coding sequencescan be cloned into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of the targetingagent/toxin coding sequences will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed (see e.g., Smith et al., JVirol 46:584 (1983); U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the targeting agent/toxin coding sequences can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing targeting agent/toxin proteins in infected hosts (see e.g.,Logan et al., Proc Natl Acad Sci USA 81:3655-3659 (1984)). Specificinitiation signals can also be required for efficient translation ofinserted targeting agent/toxin coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Exogenous translationalcontrol signals, including the ATG initiation codon, can additionallyneed to be provided. One of ordinary skill in the art would readily becapable of determining this and providing the necessary signals.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression can be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBittner et al., Methods in Enzymol 153:516-544 (1987)).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc. Forlong-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressconstructs encoding the targeting agent/toxin compounds can beengineered. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with targetingagent/toxin DNA controlled by appropriate expression control elements(e.g., promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of foreign DNA, engineered cells can be allowed to grow forone or two (1-2) days in an enriched media, and then are switched to aselective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines.

A number of selection systems can be used, including, but not limited,to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoriboxyltransferase (Szybalska etal., Proc Natl Acad Sci USA 48:2026 (1962)), and adeninephosphoribosyltransferase genes (Lowy et al., Cell 22:817 (1980)) can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., Proc NatlAcad Sci USA 77:3567 (1980); O'Hare et al., Proc Natl Acad Sci USA78:1527 (1981)); gpt, which confers resistance to mycophenolic acid(Mulligan et al., Proc Natl Acad Sci USA 78:2072 (1981)); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,J Mol Biol 150:1 (1981)); and hygro, which confers resistance tohygromycin (Santerre et al., Gene 30:147 (1984)).

After a sufficiently purified compound has been prepared, one willdesire to prepare it into a pharmaceutical composition that can beadministered parenterally. This is done by using for the lastpurification step a medium with a suitable pharmaceutical composition.

Suitable pharmaceutical compositions in accordance with the inventionwill generally comprise from about 10 to about 100 mg of the desiredconjugate admixed with an acceptable pharmaceutical diluent orexcipient, such as a sterile aqueous solution, to give a finalconcentration of about 0.25 to about 2.5 mg/mL with respect to theconjugate. Such formulations will typically include buffers such asphosphate buffered saline (PBS), or additional additives such aspharmaceutical excipients, stabilizing agents such as BSA or HSA, orsalts such as sodium chloride. For parenteral administration it isgenerally desirable to further render such compositions pharmaceuticallyacceptable by insuring their sterility, non-immunogenicity andnon-pyrogenicity. Such techniques are generally well known in the art asexemplified by Remington's Pharmaceutical Sciences, 16th Ed. MackPublishing Company (1980), incorporated herein by reference. It shouldbe appreciated that endotoxin contamination should be kept minimally ata safe level, for example, less that 0.5 ng/mg protein. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

A preferred parenteral formulation of the targeting agent/toxincompounds, including immunotoxins, in accordance with the presentinvention is 0.25 to 2.5 mg conjugate/mL in 0.15M NaCl aqueous solutionat pH 7.5 to 9.0. The preparations can be stored frozen at −10□ C to−70□ C for at least one (1) year.

G. Attachment of Other Agents to Targeting Agents

It is contemplated that most therapeutic applications of the presentinvention will involve the targeting of a toxin moiety to theendothelium, particularly tumor endothelium. This is due to the muchgreater ability of most toxins to deliver a cell killing effect ascompared to other potential agents. However, there can be circumstances,such as when the target antigen does not internalize by a routeconsistent with efficient intoxication by targeting agent/toxincompounds, such as immunotoxins, where one will desire to targetchemotherapeutic agents such as antitumor drugs, other cytokines,antimetabolites, alkylating agents, hormones, and the like. An advantageof these agents over their non-targeting agent conjugated counterpartsis the added selectivity afforded by the targeting agent, such as anantibody. Exemplary agents include, but are not limited to, such assteroids, cytosine arabinoside, methotrexate, aminopterin,anthracyclines, mitomycin C, vinca alkaloids, demecolcine, etoposide,mithramycin, and the like. This list is, of course, merely exemplary inthat the technology for attaching pharmaceutical agents to targetingagents, such as antibodies, for specific delivery to tissues is wellestablished.

It is proposed that particular benefits can be achieved through theapplication of the invention to tumor imaging. Imaging of the tumorvasculature is believed to provide a major advantage when compared topresent imaging techniques, in that the cells are readily accessible.Moreover, the technology for attaching paramagnetic, radioactive andeven fluorogenic ions to targeting agents, such as antibodies, is wellestablished. Many of these methods involve the use of a metal chelatecomplex employing, for example, an organic chelating agent such a DTPAattached to the antibody. See e.g., U.S. Pat. No. 4,472,509. In thecontext of the present invention the selected ion is thus targeted tothe tumor endothelium by the targeting agent, such as an antibody,allowing imaging to proceed by means of the attached ion.

A variety of chemotherapeutic and other pharmacologic agents have nowbeen successfully conjugated to antibodies and shown to functionpharmacologically (see e.g., Vaickus et al., Cancer Invest 9:195-209(1991)). Exemplary antineoplastic agents that have been investigatedinclude doxorubicin, daunomycin, methotrexate, vinblastine, and variousothers. Dillman et al., Antibody Immunocon Radiopharm 1:65-77 (1988);Pietersz et al., Antibody Immunoconj Radiopharm 1:79-103 (1988).Moreover, the attachment of other agents such as neocarzinostatin(Kimura et al., Immunogenetics 11:373-381 (1980)), macromycin, trenimon(Ghose et al., Meth. Enzymology 93:280-333 (1983)) and α-amanitin hasbeen described.

In addition to chemotherapeutic agents, the invention is contemplated tobe applicable to the specific delivery of a wide variety of other agentsto tumor vasculature. For example, under certain circumstances, one candesire to deliver a coagulant such as Russell's Viper Venom, activatedFactor IX, activated Factor X or thrombin to the tumor vasculature. Thiswill result in coagulation of the tumor's blood supply. One can alsoenvisage targeting a cell surface lytic agent such as phospholipase C,(Flickinger & Trost, Eur. J. Cancer 12(2):159-60 (1976)) or cobra venomfactor (CVF) (Vogel & Muller-Eberhard, Anal. Biochem 118(2):262-268(1981)) which should lyse the tumor endothelial cells directly. Theoperative attachment of such structures to targeting agents, such asantibodies, can be readily accomplished, for example, by protein-proteincoupling agents such as SMPT. Moreover, one can desire to target growthfactors, other cytokines or even bacterial endotoxin or the lipid Amoiety of bacterial endotoxin to a selected cell type, in order, e.g.,to achieve modulation of cytokine release. The attachment of suchsubstances is again well within the skill in the art as exemplified byGhose et al., CRC Critical Reviews in Therapeutic Drug Carrier Systems3:262-359 (1987).

Thus, it is generally believed to be possible to conjugate to antibodiesany pharmacologic agent that has a primary or secondary amine group,hydrazide or hydrazine group, carboxyl alcohol, phosphate, or alkylatinggroup available for binding or crosslinking to the amino acids orcarbohydrate groups of the antibody. In the case of protein structures,this is most readily achieved by means of a cross linking agent asdescribed above. In the case of doxorubicin and daunomycin, attachmentcan be achieved by means of an acid labile acyl hydrazone or cisaconityl linkage between the drug and the antibody. Finally, in the caseof methotrexate or aminopterin, attachment is achieved through a peptidespacer such as L-Leu-L-Ala-L-Leu-L-Ala (SEQ ID NO: 2), between theγ-carboxyl group of the drug and an amino acid of the antibody.

Alternatively, any such structures which are nucleic acid-encodedstructures can be operatively attached to the targeting agents of theinvention by standard recombinant DNA techniques, such as, for example,those discussed above.

EXAMPLES

The following Examples have been included to illustrate preferred modesof the invention. Certain aspects of the following Examples aredescribed in terms of techniques and procedures found or contemplated bythe present inventors to work well in the practice of the invention.These Examples are exemplified through the use of standard laboratorypractices of the inventors. In light of the present disclosure and thegeneral level of skill in the art, those of skill will appreciate thatthe following Examples are intended to be exemplary only and thatnumerous changes, modifications and alterations can be employed withoutdeparting from the spirit and scope of the invention.

Example 1 Endothelial Localization of Receptor Tyrosine Phosphatase,ECRTP/DEP-1, in Developing and Mature Renal Vasculature

Developmental assembly of the renal microvasculature is a preciseprocess requiring spatially and temporally coordinated migration,assembly, differentiation and maturation of endothelial cells in thecontext of adjacent epithelial and mesangial cells. Moleculardeterminants of assembly are largely undefined, yet requirements forcell surface receptors to direct context appropriate endothelialresponses are anticipated. Endothelial expression and distribution ofthe receptor tyrosine phosphatase, ECRTP/DEP-1, were evaluated duringdevelopmental assembly of the renal microvasculature. Monoclonalantibodies generated against ECRTP/DEP-1 ectodomain epitopes localizeits expression to membrane surfaces of endothelial cells in glomerular,peritubular capillary and arterial renal circulations of mature humanand murine kidney. During kidney development, ECRTP/DEP-1 immunostainingis evident on a subpopulation of metanephric mesenchymal cells and onputative progenitors of glomerular capillary endothelial cells early intheir recruitment to developing glomeruli. ECRTP/DEP-1 is prominentlydisplayed on luminal membrane surfaces with punctate accumulations atinter-endothelial contacts that overlap, but do not co-localize with VEcadherin. In vitro studies show that ECRTP/DEP-1 is recruited topositions of inter-endothelial contact in confluent cultured human renaland dermal microvascular endothelial cells, where its distributionoverlaps, but again does not coincide with VE cadherin. Experimentaldissociation of VE cadherin from endothelial junctional complexes doesnot redistribute ECRTP/DEP-1 away from inter-endothelial contacts. Thesefindings indicate that ECRTP/DEP-1 ectodomains interact with proteinsthat are expressed on surfaces of endothelial cells and that are engagedby cell-cell contact, to convey signals for cell recognition, or arrestof migration or proliferation.

In order to identify receptor tyrosine phosphatases expressed in humanrenal microvascular endothelial cells (HRMEC), degenerateoligonucleotide primers derived from conserved phosphatase domains wereused to amplify and sequence cDNAs representing expressed messages,according to methods described in Schoecklmann et al., J Am Soc Nephrol5:730 (1994)(abstract). Among putative receptor cDNAs identified was onethat was designated ECRTP (endothelial cell receptor tyrosinephosphatase), a product virtually identical to the DEP-1 (for densityenhanced phosphatase) cDNA cloned by Ostman et al. from HeLa cells andregulated in abundance by cell density in WI-38 cells. Ostman et al.,Proc Natl Acad Sci USA 91:9680-9684 (1994). ECRTP/DEP-1 (also calledbyp-1, HPTPη, and CD148) expression has been identified in neonatalsmooth muscle cells, in breast and thryoid cancer cell lines, and in allhematopoietic lineages. Keane et al., Cancer Research 56:4236-4243(1996); de la Fuente-Garcia et al., Blood 91:2800-2809 (1998). AlthoughECRTP/DEP-1 expression was identified in arterial endothelial cells ofthe kidney, in situ hybridization experiments failed to detectglomerular capillary localization of ECRTP/DEP-1mRNA. Borges et al.,Circulation Research 79:570-580 (1996). The developmental timing anddistribution of its expression have not been previously reported.

Like other members of the Class III receptor tyrosine phosphatasefamily, including GLEPP-1, SAP-1, and DPTP 10D, ECRTP/DEP-1 is a type Imembrane protein characterized by a large extracellular domaincontaining eight or more fibronectin type III repeats and a singlecytoplasmic domain phosphatase catalytic domain. Ostman et al., ProcNatl Acad Sci USA 91:9680-9684 (1994). The GLEPP-1 receptor tyrosinephosphatase is structurally similar to ECRTP/DEP-1, yet shows renalexpression limited to glomerular visceral epithelial cells, where it hasbeen implicated in podocyte integrity. Thomas et al., J Biol Chem269:19953-19962 (1994). Unlike the MAM domain containing receptors, PTPμκ and λ, available data do not support participation of class IIIreceptors in homophilic binding, and ligands have not yet beenidentified.

Monoclonal antibodies were developed against ECRTP/DEP-1 ectodomainepitopes to characterize its distribution in the renal circulation ofmature and developing kidney. ECRTP/DEP-1 is expressed at high levels inglomerular, peritubular and renal arterial endothelial cells and shows apattern of distribution in vivo and in vitro that suggests itcontributes to cell-cell recognition required for capillary assembly andmaintenance.

Methods

Cell lines and cell culture—Primary human renal microvascularendothelial cells (HRMEC) were isolated, cultured, and used at third orfourth passage after thawing, as described. Martin et al., In Vitro CellDev Biol 33:261-269 (1997). Human dermal microvascular endothelial cells(HMEC-1 cells, CDC) were grown in MCDB131 media (Sigma Chemical Co. ofSt. Louis, Mo.) containing 15% fetal bovine serum (Hyclone Laboratories,Logan Utah, USA), 10 ng/ml epidermal growth factor (CollaborativeBiomedical Products, Becton Dickinson, Bedford, Mass.), and 1 mg/mlhydrocortisone (Sigma Chemical Co. of St. Louis, Mo.) Ades et al., JInvest Dermatol. 99:683-690 (1992). Madin Darby Canine Kidney (MDCK)cells (kindly provided by L. Limbird, Vanderbilt Pharmacology) weregrown in Dulbecco's minimal essential medium (DMEM, GIBCO BRL,Rockville, Md.) containing 4.5% D-glucose and supplemented with 10%fetal bovine serum. All growth medium was supplemented with 1 mML-glutamine (GIBCO BRL, Rockville, Md.), 100 units/ml penicillin and 100mg/ml streptomycin (GIBCO BRL, Rockville, Md.).

Generation of antibodies to recombinant ECRTP/DEP-1 proteins—Ectodomain(amino acids 175-536) and catalytic domain (amino acids 1048-1338)sequences of human, ECRTP/DEP-1 (SEQ ID Nos: 3 and 4; Ostman et al.,Proc Natl Acad Sci USA 91:9680-9684 (1994)), were subcloned into thepRSET vector (Invitrogen, Carlsbad, Calif.). Recombinant fusion proteinswere expressed in bacteria, purified by a kit sold under the registeredtrademark NI-AGAROSE AFFINITY™ by Invitrogen of Carlsbad, Calif., andcharacterized by SDS-PAGE as greater than 95% homogeneous proteins of 40and 36 kDa, respectively. Mouse hybridoma antibodies (ECRTP-Ab1,ECRTP-Ab2) were generated against ECRTP/DEP-1 ectodomain(ECRTP/DEP-1_(ec)) protein by intra-peritoneal immunization, fusion withSP2-0 cells, ELISA screening, selection, expansion and purification byaffinity chromatography on PROTEIN A-AGAROSE (Sigma Chemical Co. of St.Louis, Mo.).

Immunodetection of exogeneously expressed ECRTP/DEP-1—MDCK cells grownin 100 mm plastic dishes (sold under the registered trademark FALCON® byBecton, Dickinson and Company, Franklin Lakes, N.J.) were transfectedwith an expression plasmid pSRα DEP-1/3xHA that drives high levelexpression of the human ECRTP/DEP-1 receptor modified by addition ofthree repeats of a hemagglutinin peptide (HA) to the carboxy terminus,using cationic lipid (LIPOFECTAMINE™, GIBCO BRL, Rockville, Md.)according to the manufacturer's protocol. Forty eight hours aftertransfection, cells were placed on ice, washed twice with ice coldPBS(−) and immediately lysed in 0.5 ml lysis buffer (50 mM HEPES pH 7.5,50 mM NaCl, 5 mM EDTA, 2 μg/mL aprotinin, 1 μg/mL leupeptin, 1 mM PMSF).Lysates were clarified by centrifugation, and membrane receptors wererecovered by batch adsorption to WGA-Agarose (Sigma Chemical Co. of St.Louis, Mo.) for 4 hours at 4⁰ C. The resultant precipitates wereresolved by 7% SDS-PAGE under reducing conditions, transferred toImmobilon-P transfer membranes (Millipore Corporation, Bedford, Mass.),and blocked in 5% non-fat dry milk in Tris-buffered saline (50 mM TrisHCl pH 7.5, 137 mM NaCl) containing 0.2% Tween 20 (TBST) overnight at 4°C. Blots were incubated with murine monoclonal ECRTPAbs 1 or 2 (10μg/mL) or anti-HA (2.5 μg/mL) antibody followed by incubation withhorseradish peroxidase-conjugated rabbit anti-mouse IgG antibody(Boehringer Mannheim, Indianapolis, Ind.). Membranes were washed withTBST, then developed using a chemiluminescent substrate (ECL, Amersham,Buckinghamshire, England) according to the manufacturer's instructions.

Generation of Stably Transfected MDCK Cells and Cell Staining—MDCK cellswere transfected with an expression plasmid pCDNA3 DEP-1/3xHA(Invitrogen) using cationic lipids (Lipofectamine™, GIBCO BRL,Rockville, Md.) according to the manufacturer's protocol. Stabletransfectants were selected by addition of G418 (GIBCO BRL, Rockville,Md.) to culture media at a final concentration of 800 μg/mL, and asingle colony was obtained by limited dilution cloning. The cells weregrown on glass coverslips (Fisher Scientific, Pittsburgh, Pa.) and fixedwith 100% methanol for 10 min at −20-C. Coverslips were washed withphosphate buffered saline, blocked with 5% goat serum for 30 min at roomtemperature, incubated with ECRTPAb-2 (10 μg/mL) for 60 min, washed,then incubated with FITC conjugated goat anti-mouse IgG (JacksonImmunoresearch Laboratory, Westgrove, Pa.) for 60 min. Coverslips weremounted and analyzed by confocal microscopy (equipment sold under theregistered trademark ZEISS® LSM410™ by Zeiss, Oberkochen, Germany). Topreabsorb the immunoreactivity of ECRTP/DEP-1-Ab, 50 μg of ECRTP/DEP-1proteins (Ec or Cy) were preincubated with ECRTPAb-2 for 4 hours at 4-C,microcentrifuged at 15,000 rpm for 20 min and the resultant supernatantwas used to stain cells.

Tissue immunolocalization—Human kidney tissue was snap-frozen in a dryice-acetone bath. Cryostat sections (4 mm) were fixed in acetone at −20°C. for 10 min, washed with phosphate buffered saline, and pre-adsorbedwith avidin-biotin blocking reagents (Vector Laboratories, Inc. ofBurlingame, Calif.) according to manufacturer's instructions. Sectionswere washed with phosphate buffered saline, blocked with 5% goat serum,incubated with monoclonal ECRTP/DEP-1 antibody (ECRTP-Ab1, 10 μg/mL, 10min), washed, incubated with biotinylated goat anti-mouse IgG (VectorLaboratories, Inc. of Burlingame, Calif., 7.5 μg/mL, 60 min), washed,incubated with fluorescein isothiocyanate (FITC)-conjugated streptavidin(Pierce Chemical Company of Rockford, Ill., 4 μg/ml, 30 min) and finallywashed with phosphate buffered saline. Coverslips were mounted (soldunder the trademark VECTASHIELD™ by Vector Laboratories, Inc. ofBurlingame, Calif.) and analyzed by confocal microscopy (equipment soldunder the registered trademark ZEISS® LSM410™ by Zeiss, Oberkochen,Germany). For colocalization experiments, acetone fixed frozen sectionswere blocked with 5% donkey serum, and incubated with mixture ofECRTP/DEP-1 antibody (10 μg/mL) and goat VE cadherin antibody (5 μg/mL,Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) at room temperaturefor 60 minutes. Specific antibodies were detected using a mixture ofFITC-conjugated donkey anti-mouse and rhodamine conjugated donkeyanti-goat antibodies (Jackson Immunoresearch Laboratory, Westgrove, Pa.)at room temperature for 60 minutes. Specific immunostaining for eachantigen was identified in overlapping images generated by analysis ofthe same section at 488 nm and 568 nm wavelengths, respectively, on aZEISS® LSM410™ confocal microscope.

Immunolabeled murine kidney sections showed high background and requiredan alternative technique. The anti-ECRTP/DEP-1 mAb, ECRTP-Ab1, wasdirectly coupled to FITC. Briefly, ECRTP-Ab1 (0.55 mL of 0.94 mg IgG/mLin 0.1 M sodium carbonate buffer, pH 9.0) was conjugated to 0.03 mL FITCsolution (Sigma Chemical Co. of St. Louis, Mo., 1.0 mg/ml in DMSO)overnight at 4° C. The reaction was stopped by adding ammonium chlorideto 50 mM final concentration. Following incubation for 2 hours at 4° C.,the mixture was dialyzed exhaustively against phosphate buffered salineto remove unbound FITC. A mouse monoclonal IgG against rat glomerularbasement membrane coupled to FITC using the identical protocol was usedas a control. Hyink et al., Am J Physiol 270:F886-F899 (1996). Acetonefixed sections were blocked with 0.5 M ammonium chloride, incubated for30 min with MoAb-FITC conjugates, washed, and examined byepifluorescence microscopy. In some additional control experiments, theanti-DEP-FITC conjugate was mixed with a molar excess of theimmunization peptide before incubation with the sections.

Immunoblots and immunocytochemistry of human endothelial cell linesHuman endothelial cells grown in 60 mm dishes were lysed at confluencyin 0.5 mL of lysis buffer (20 mM TrisCl pH7.5, 50 mM NaCl, 1 mM EDTA,0.5% Triton X-100, 0.5% SDS, 0.5% deoxycholate, 2 μg/mL aprotinin, 1μg/mL leupeptin, 1 mM phenylmethylsulfonylfluoride) on ice for 30minutes. Cleared lysate protein, 150 μg, was incubated with 10 μg/mL ofaffinity purified rabbit ECRTP/DEP-1 antibody or rabbit IgG (SigmaChemical Co. of St. Louis, Mo.) at 4° C. for 4 hours, andimmunoprecipitates were recovered using Protein-a Sepharose (SigmaChemical Co. of St. Louis, Mo.). SDS-PAGE, and immunoblotting procedureswere carried out as described above. Endothelial cells were grown onuncoated glass coverslips (Fisher Scientific, Pittsburgh, Pa.), thenfixed with 50% methanol for 10 min at 4° C. Coverslips were washed withPBS, blocked with 5% goat serum for 30 min at room temperature,incubated with ECRTPAb-2 monoclonal antibody (10 μg/ml) or VE cadherinmonoclonal antibody (2 μg/mL, BD Transduction Laboratory, Lexington,Ky.) for 60 min, washed, then incubated with biotinylated goatanti-mouse IgG (Vector Laboratories of Burlingame, Calif.) for 60 min,washed, and finally incubated with fluorescein conjugated (FITC)streptavidin (4 μg/ml, Pierce Chemical Company of Rockford, Ill.) for 30min. Coverslips were mounted and analyzed by confocal microscopy(equipment sold under the registered trademark ZEISS® LSM410™ by Zeiss,Oberkochen, Germany).

Calcium chelation to disrupt inter-endothelial cadherincomplexes—Confluent HMEC-1 cells grown on glass coverslips in DMEM mediasupplemented with 15% fetal bovine serum were exposed to addition ofEGTA (ethylene glycol-bis(b-aminoethylether)-N,N,N′,N′,-tetraaceticacid, Sigma Chemical Co. of St. Louis, Mo.) to reach a finalconcentration of 5 mM. Cells were incubated for an additional 20 min,then fixed with 50% methanol at 4° C. for 10 min, washed with phosphatebuffered saline, and stained with monoclonal ECRTP/DEP-1 antibody (10μg/ml) or VE cadherin monoclonal antibody (2 μg/mL, BD TransductionLaboratory, Lexington, Ky.), as described above.

RESULTS—Monoclonal antibodies recognize recombinant and expressedECRTP/DEP-1. Recombinant fusion proteins representing either ectodomain(Ec) or cytoplasmic domain (Cy) ECRTP/DEP-1 sequences were expressed inbacteria and used to immunize rabbits and/or mice. Shown in FIG. 1A,monoclonal antibodies, ECRTPAb-1 and ECRTPAb-2, specifically identifythe ectodomain but not the cytoplasmic domain recombinant proteins. Toascertain whether these antibodies recognize the full length proteinexpressed in mammalian cells, MDCK cells were transiently transfectedwith either an empty expression plasmid (SRa) or one driving expressionof a full length ECRTP/DEP-1 tagged on the carboxy terminus with ahemagglutinin epitope (SRa DEP-1/HA). Cell lysates from transfectedcells were immunoprecipitated using the epitope-specific monoclonalanti-HA antibody, then probed with the antibodies indicated, includingECRTPAb-1 and ECRTPAb-2 (FIG. 1B). Both recognized the 220 kDa HA-taggedECRTP/DEP-1.

Finally, capacity of the monoclonal antibodies to specifically recognizethe ECRTP/DEP-1 expressed in intact cells was assessed using MDCK cellsstably transfected with ECRTP/DEP-1. Indirect epifluorescence stainingwith ECRTPAb-2 localized ECRTP/DEP-1 to lateral cell membranes (FIG. 1C,Panel a), a finding confirmed in confocal Z plane sections of MDCK cellsgrown to confluence on permeable membrane supports. Competition with theimmunizing peptide (Ec) blocked immunostaining (FIG. 1C, Panel c) whilethe irrelevant cytoplasmic domain fusion peptide (Cy) did not (FIG. 1C,Panel d). ECRTP/DEP-1 immunoreactivity localizes to endothelial cells ofglomerular capillaries, peritubular capillaries and renal arteries. Todetermine the distribution of ECRTP/DEP-1 in mature mammalian kidney,indirect or direct immunofluorescence staining experiments wereconducted on frozen sections from human and mouse sources. Shown in FIG.2, ECRTP-Ab2 immunolocalizes ECRTP/DEP-1 expression to arterial,glomerular and peritubular capillaries, and in particular, to theendothelial cells in these sites. Higher magnification frames showpredominant ECRTP/DEP-1 labeling along the luminal membranes ofendothelial cells, at least in the arterial sites where endothelialmembrane definition is most reliable (FIG. 3).

The punctate characteristic of the staining in the glomerularmicrocirculation led to the evaluation of whether ECRTP/DEP-1 wasengaged in inter-endothelial junctional complexes. In double labelingstudies using ECRTP-Ab1 and VE-cadherin antibodies, some overlap wasevident (FIG. 3). In addition to the luminal endothelial membranestaining, a regional accumulation of ECRTP/DEP-1 was evident at pointsof inter-endothelial contact, overlapping, but not limited, to theendothelial junctional complexes that include VE cadherin. Lampugnani etal., J Cell Biol 129:203-217 (1995). This pattern was evident in botharterial and peritubular capillaries. In extra-renal sites, capillaryand large vessel endothelial cells of brain, lung, liver and spleen wasidentified and endocardial staining were also apparent.

Based on the prominent ECRTP/DEP-1 expression in vascular endothelium ofmature kidney, temporal and spatial expression of this receptor duringrenal vascular development in mouse embryos was evaluated. Shown in FIG.4, ECRTPAb-1 binds as an antigen, its murine ECRTP/DEP-1, based on itssimilar pattern of staining is mature murine and human kidneys, andbased on the effect of the recombinant human immunogen (Ec) to blockstaining of the mouse tissue. In developing mouse kidneys at E14, E16,and postnatal day 6, (FIG. 4A-C) conjugates of ECRTP-Ab1-FITC displayeda pattern of immunoreactivity that was strikingly similar to the patternobserved previously using antibodies against the VEGF receptor, flk-1,and the EphB1/ephrin-B1 receptor-ligand. Daniel et al., Kidney Int50:S-73-S-81 (1996). Notably, ECRTP-Ab1-FITC bound to endothelial cellsof developing glomeruli and microvessels in the fetal kidney cortex.Small but intense foci of bound antibody were observed on isolatedcortical mesenchymal cells believed to be angioblasts (FIGS. 4A & 4B).Within vascular clefts of comma- and S-shaped developing glomeruli, asubpopulation of cells consistent with glomerular endothelial precursorswere labeled (FIGS. 4A & 4B).

Immunolabeling for ECRTP/DEP-1 on sections of neonatal kidney produced adistinct vascular labeling pattern (FIG. 4C). Arteriolar, glomerular,and peritubular capillary endothelia all labeled intensely (FIG. 3C).Glomerular endothelial cells were also brightly labeled in adult mousekidney (FIG. 4D), as they were in sections of human kidney. Other cellswithin the immature and mature kidneys did not bind ECRTP-Ab1-FITC, andsections labeled with control monoclonal IgG-FITC conjugates, ormixtures of ECRTP-Ab1-FITC and the immunization peptide (Ec) showed nostaining.

Independent immunoblot and immunofluorescence staining experiments usingECRTP-Ab1 showed high level expression in endothelial cells culturedfrom a range of different vascular sites, including the HRMEC from whichit was cloned, a dermal microvascular endothelial cell line, HMEC-1(Ades et al., J Invest Dermatol 99:683-690 (1992); human umbilical veinendothelial cells; and a HUVEC derived cell line, Eahy926 (Bauer et al.,J Cell Physiol 153:437-449 (1992). Epitopes recognized by this antibodywere not detected in non-endothelial cell lines; including HEK293 cells,glomerular mesangial cells, vascular smooth muscle cells, and P19embryonic carcinoma cells.

Shown in FIG. 5 are patterns of ECRTP/DEP-1 localization in human renalmicrovascular endothelial cells, HRMEC (Panel A), and human dermalmicrovascular endothelial cells, HMEC (Panel B). Confluent HRMECcultures displayed prominent staining with ECRTP-Ab2 at points ofinter-endothelial contact. In addition, there were punctateaccumulations of apical membrane staining in confocal planes capturingthe apical surface (Panel A), but not on the basal membrane surface.Endothelial cells plated at sufficiently low density to be isolated fromcontact with one another did not show the prominent pattern of cellborder staining seen in contacting cells. It should be noted thatECRTP-Ab1 did not demonstrate the inter-endothelial localization seenwith ECRPT-Ab2, but stained only the subpopulation of receptors evidenton the apical surface.

This apparent accumulation of ECRTP/DEP-1 at sites of endothelialcell-cell contact is consistent with the punctate accumulations ofstaining seen in intact mature vessels, and suggests that asubpopulation of receptors distribute to points of inter-endothelialcontact. Thus, the distribution of ECRTP/DEP-1 was compared with that ofVE cadherin. Confocal localization of ECRTP/DEP-1 and VE cadherinimmunoreactivity in double labeling experiments of confluent HMECcultures again showed modest overlap of ECRTP/DEP-1 staining with theVE-cadherin localized in inter-endothelial junctions. Similar patternsof colocalization were seen in double labeled sections of human kidneytissue (FIG. 3). Finally, experiments were conducted to ascertainwhether the intercellular accumulation of ECRTP/DEP-1 immunoreactivityrequired the integrity of VE-cadherin interactions. Shown in FIG. 5B,EGTA treatment of the HMEC-1 cells dissociates VE cadherin from theinter-endothelial junctional complexes, but has no apparent effect onECRTP/DEP-1 localization over the 20-30 minute time period of theexperiment. This result suggests that any inter-endothelial junctionsthat can retain ECRTP/DEP-1 do not require cadherin integrity.Furthermore, these data are consistent with the observations thatECRTP/DEP-1 and VE cadherin overlap, but do not co-localize precisely inintact vessel endothelium (FIG. 3).

DISCUSSION—Several of the observations presented here provide newinsights about the ECRTP/DEP-1 tyrosine phosphatase in vasculardevelopment and in endothelial cell-cell interactions. The significanceof the initial identification of ECRTP/DEP-1 as a transcript expressedin cultured human renal microvascular endothelial cells has beenconfirmed at several levels. Schoecklmann et al., J Am Soc Nephrol 5:730(1994)(abstract). Cultured HRMEC's express the protein on cellmembranes, just as glomerular and peritubular capillaries do in intactkidney tissue. Indeed, capillary and arterial endothelium appear to bethe dominant cellular sources of ECRTP/DEP-1 expression in mature humanand mouse kidney. In contrast with the previous in situ experiments inrat kidney kidneys, described in Borges et al., Circulation Research79:570-580 (1996), high level expression were found in glomeruli of bothmouse and human.

Careful evaluation of the sites of membrane to which ECRTP/DEP-1distributes has shown prominent apical membrane staining in arterialendothelium in addition to the inter-endothelial membrane staining thatappears responsible for the somewhat granular staining pattern in theglomerular capillaries. The lateral cell membrane distribution ofECRTP/DEP-1 in the artificial MDCK epithelial cell system and incontacting cultured HRMEC (FIG. 5), led to the formally evaluation ofthe relationship of lateral ECRTP/DEP-1 membrane accumulation with VEcadherin complex integrity. The in situ overlap of ECRTP/DEP-1 and VEcadherin immunostaining is modest (FIG. 3), and is restricted to veryfocal regions of inter-endothelial contact in some, but not alljunctional complexes. As ECRTP/DEP-1 lateral membrane distribution ismaintained in cultured endothelial cells in which VE cadherin complexeshave been dissociated by calcium chelation, it is concluded that thereis neither anatomical co-localization nor functional correlation ofECRTP/DEP-1 distribution with maintenance of inter-endothelialcomplexes. These findings, however, cannot exclude the possibility thatlateral ECRTP/DEP-1 membrane distribution can function to establishconditions permissive to assembly of inter-endothelial complexescontaining VE cadherin.

Alternatively, the lateral membrane distribution can reflect interactionof the ECRTP/DEP-1 extracellular domain with a putative ligand expressedon contacting membranes that is capable of redistributing receptors orstabilizing them in ligand-receptor complexes created through juxtacrineengagement. Certainly there is available evidence that membraneassociated receptor tyrosine phosphatase activity is increased incultured cells, including endothelial cells, that are in close contact.Pallen and Tong, Proc Natl Acad Sci USA 88:6996-7000 (1991); Batt etal., J Biol Chem 273:3408-3414 (1998). In the culture systems presentedin this Example, an increase in ECRTP/DEP-1 activity that correlateswith cell density and with cell-density mediated growth arrest has beendemonstrated.

The apical membrane distribution of ECRTP/DEP-1 in arterial andapparently in capillary endothelium is intriguing, particularly in thecontext of data showing that platelets and all hematopoietic lineagesexpress the ECRTP/DEP-1. Palou et al., Immunol Lett 57:101-103 (1997).Homophilic interactions between ECRTP/DEP-1s of endothelial cells andcirculating cells that can encounter them on luminal membranes of intactvessels suggest that it is likely that regulatory factors orco-receptors on each of the engaging cells are important in modulatingany downstream responses.

Finally, the data assessing the developmental pattern of ECRTP/DEP-1expression on cells that contribute to assembly of the glomerularcapillary network offers insight about roles for this receptor in thiscoordinated process. Receptor tyrosine phosphatases of the ECRTP/DEP-1subclass, including DPTP10D, have been assigned important roles in thetargeting of neurons to correct destinations during development. Desaiet al., Cell 84:599-609 (1996). Previous reports have identifiedexpression in hematopoietic progenitors, including erythroid, lymphoidand myeloid series lineages. Palou et al., Immunol Lett. 57:101-103(1997). With the accumulating evidence that hemangioblasts serve ascommon precursors of both hematopoietic and vascular endotheliallineages, it now appears that ECRTP/DEP-1 expression is initiated earlyin the ontogeny of these precursors. Furthermore, it appears thatECRTP/DEP-1 can function to promote differentiation of erythroid lineagecells that express it. Kumet et al., J Biol Chem. 271:30916-30921(1996).

Example 2 ECRTP/DEP-1 Mediates Signals for Endothelial Growth Arrest andMigration Inhibition

Powerful endogenous inhibitors of angiogenesis, such as thrombospondin,angiostatin and endostatin, inhibit the proliferation and migration ofcultured endothelial cells in vitro. Such angiogenesis inhibitorycontrols appear to signal arrest of endothelial growth and migration byengaging endothelial surface receptors. One of the most powerful growthinhibitory signals for cultured endothelial cells is imposed bycell-cell contact, which is described in the art as “density mediatedgrowth arrest” or “contact mediated growth arrest”. High levelexpression of the receptor tyrosine phosphatase, ECRTP/DEP-1, atinter-endothelial contacts in microvascular and large vessel endotheliumof human kidney and other organs is described in Example 1.

In this Example, the ECRTP/DEP-1 has been determined to mediateendothelial growth and migration arrest signals. The ECRTP/DEP-1 iscatalytically activated in conjunction with cell-cell contact. Transientoverexpression of full length ECRTP/DEP-1 arrests endothelial growth andmigration. Bivalent forms of a monoclonal antibody, ECRTPAb-1, thatbinds the ECRTP/DEP-1 ectodomain inhibits endothelial proliferation andmigration, while Fab fragments are inactive. This antibody imposesinhibition on corneal angiogenic responses in a mouse system. Thesefindings indicate that the ECRTP/DEP-1 signals endothelial growth andmigration arrest upon engagement of its ligand on the surfaces ofcontacting endothelial cells, and that surrogate activators, ormodulators, of endothelial growth arrest signals are viable candidatesfor angiogenesis inhibitors.

Methods

Cell Culture—Primary human renal microvascular endothelial cells, HRMEC,were isolated, cultured, and used at third or fourth passage afterthawing, as described in Martin et al., In Vitro Cell Dev Biol33:261-269 (1997). Human dermal microvascular endothelial cells (HMEC-1cells, CDC) were grown in MCDB131 media (Sigma Chemical Co. of St.Louis, Mo.) containing 15% fetal bovine serum (Hyclone Laboratories,Logan Utah, USA), 10 ng/mL epidermal growth factor (CollaborativeBiomedical Products; Becton Dickinson, Bedford, Mass.), and 1 μg/mLhydrocortisone (Sigma Chemical Co. of St. Louis, Mo.). Ades et al., JInvest Dermatol 99:683-690 (1992). All growth media were supplementedwith 1 mM L-glutamine (GIBCO BRL, Rockville, Md.), 100 units/mLpenicillin and 100 □g/mL streptomycin (GIBCO BRL, Rockville, Md.).

Antibodies—Ectodomain (ECRTP/DEP-1_(ec), amino acids 175-536) andcatalytic domain (ECRTP/DEP-1_(cy), amino acids 1048-1338) sequences ofhuman ECRTP/DEP-1 (SEQ ID Nos:3 and 4; Ostman et al., Proc Natl Acad SciUSA 91:9680-9684 (1994)) were subcloned into the pRSET vector(Invitrogen, Carlsbad, Calif.). Recombinant fusion proteins wereexpressed in bacteria, purified by Ni-agarose affinity (Invitrogen,Carlsbad, Calif.), and characterized by SDS-PAGE as greater than 95%homogeneous proteins of 40 and 36 kDa, respectively. Rabbit antiserum tothe ECRTP/DEP-1_(cy) protein was generated by repetitive immunization,and was affinity purified, as described in Koenig et al., J Clin Immunol13:204-211 (1993). Mouse hybridoma antibody ECRTPAb-1 was obtainedfollowing immunization with ECRTP/DEP-lec protein by intraperitonealimmunization, fusion with SP2-0 cells, ELISA screening, selection,expansion and purification by affinity chromatography on proteinA-agarose (Sigma Chemical Co. of St. Louis, Mo.).

Assays for ECRTP/DEP-1 Abundance and Tyrosine Phosphatase Activity—Cellsplated at the densities and harvested at the times indicated in theFigure Descriptions were washed repeatedly with iced phosphate bufferedsaline before in situ addition of 2 ml of buffer containing 50 mM Hepes(pH 7.5), 50 mM NaCl, 5 mM EDTA, 1 mM PMSF, 1 mM β-mercaptoethanol, 1%Triton X-100. Detergent solubilized cells were incubated for 15 min at4[ ]C and insoluble material was removed by repeated microcentrifugation(two times) at 13,000×g, 10 min, 4[ ]C. Proteins in solubilizedfractions were quantitated using a modified BCA assay (Pierce ChemicalCompany of Rockford, Ill.). In some experiments, batch adsorption andelution from Triticum vulaaris lectin (WGA) conjugated to agarose (SigmaChemical Co. of St. Louis, Mo.) was conducted as described in Stein etal., J Biol Chem 271:23588-23593 (1996). Final elution for fractionssubjected to phosphatase assays was in buffer containing 25 mM imidazole(pH 7.2), 0.1 mg/ml bovine albumin, 10 mM dithiothreitol (phosphataseassay buffer), plus 3 mM N,N′,N″ triacetylchitotriose (Sigma ChemicalCo. of St. Louis, Mo.). ³²P-labeled, phosphorylated substrate (Raytide)was prepared by the manufacturer's recommendations as described(Oncogene Sciences of Uniondale, N.Y.) to achieve specific activities of(dpm/fmol). Phosphatase activity in lectin purified fractions wasassayed in triplicate at 30° C. for times indicated in the FigureDescriptions in 200%1 volumes of phosphatase buffer using 300 ng/mlsubstrate in the presence or absence of Na₃VO₄, as described. Releasedphosphate was quantitated by scintillation counting and data areexpressed as mean cpm +/−SEM. Assays were linear over 1-10 min periods.

For determination of ECRTP/DEP-1 activity and abundance (FIG. 8) HRMECcells were plated at cell densities indicated in the FigureDescriptions. At 36 hours after plating, a subset of cells, asindicated, was treated for 10 min with pervanadate (1 mM H₂O₂+1 mMNa₃VO₄), then cells were lysed in buffer containing 50 mM HEPES/pH7.5,150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1 mM, 5 μg/ml aprotinin, 1μg/ml leupeptin, 1 mM PMSF, clarified by centrifugation and equivalentlysate proteins (150 μg) were immunoprecipitated by incubation withaffinity-purified monospecific ECRTP/DEP-1 rabbit antibody (12.5 μg/mL)overnight at 4° C., and collected on protein A-sepharose (Sigma ChemicalCo. of St. Louis, Mo.).

The washed immunocomplexes were assayed for PTP activity withp-nitrophenylphosphate, pNPP (Sigma Chemical Co. of St. Louis, Mo.) aspreviously described in Wang, Y. and Pallen, C. J., J Biol Chem267:16696-16702 (1992). Briefly, the immunocomplexes were incubated withreaction mixture (50 mM sodium acetate/pH5.5, 0.5 mg/mL bovine albumin,0.5 mM DTT, 5 mM pNPP) at 30° C. for 30 min in the absence or presenceof 1 mM Na₃VO₄. Reactions were stopped by addition of 2N NaOH, and theabsorbance at 410 nm was measured.

For quantification of ECRTP/DEP-1 abundance, immunoprecipitatedfractions were also resolved by 7% SDS-PAGE under reducing conditions,transferred to PVDF membranes (sold under the trademark IMMOBILON-P™ byMillipore Corporation, Bedford, Mass.), and blocked in 5% non-fat drymilk in Tris-buffered saline (50 mM Tris/HCl pH7.5, 137 mM NaCl)containing 0.2% Tween 20 (TBST) overnight at 4° C. Blots were incubatedwith ECRTPAb1 (10 □g/mL) or phosphotyrosine monoclonal antibody, 4G10,(1.0 μg/mL, Upstate Biotechnology) and bound antibodies detected withhorseradish peroxidase-conjugated rabbit anti-mouse IgG antibody(Boehringer) and a chemiluminescent reagent (ECL; Amersham,Buckinghamshire, England).

Proliferation Assays—In initial assays of HRMEC proliferation (FIG. 6),cells were plated at the indicated density, harvested at the indicatedtimes and counted in quintriplicate. Data represent means±SEM. In otherexperiments (FIGS. 8 and 9), HMEC-1 cells were grown on a 35 mm diameterdish (sold under the registered trademark FALCON® by Becton, Dickinsonand Company, Franklin Lakes, N.J.) and cotransfected with ECRTP/DEP-1expression plasmids (either parent vector, pSRα, orpSRα-ECRTP/DEP-1/3xHA, driving high level expression of acarboxyterminal hemagglutinin (HA) epitope tagged human ECRTP/DEP-1, 1.8μg) and a green fluorescence protein expression plasmid (pEGFP,Clontech, 0.4 μg). An adenovirus-assisted lipofectamine procedure thattransfects 40-50% of HMEC-1 cells under these conditions was used, as isalso described in Example 1. Transfected cells were harvested 48 h aftertransfection and replated on glass coverslips in individual wells of a12 well plate at densities indicated in the Figure Descriptions (range2-10×10⁴), to achieve attached cell confluencies of 20-90+%).Proliferating cells were labeled by addition of 10[ ]M5-Bromo-2′-deoxy-uridine (BrdU) to culture media for 30 min at 70 hoursafter transfection. BrdU incorporation was immunocytochemically detectedusing a monoclonal BrdU antibody and rhodamine-conjugated anti-mouseIgG, according to manufacturer's protocol (Boehringer Mannheim,Indianapolis, Ind.). The cells of at least five independent fields wereobserved under epifluorescence microscopy (Nikon ECLIPSE® E600™) and thefrequency of BrdU labeling in GFP positive cells was scored.

Planar Endothelial Migration Assay—A planar endothelial migration assaywas developed to assess the rate of endothelial closure of circular“wounds” of 300-500□ diameter. A rotating silicon-tipped bit attached toa drill press was used to generate 3-5 “wounds” in confluent endothelialmonolayers within individual wells of multi-well plates. At the time of“wounding”, medium in individual wells were supplemented with agents atconcentrations indicated in the Figure Descriptions. Residual areas ofindividual wounds in photomicroscopic images captured at the indicatedtimes (4 & 8 hours) were quantitated using a Bioquant (Nashville, Tenn.)software package calibrated to a Nikon DIAPHOT® microscope. Expressed inthis manner, the rates of wound closure are remarkably linear, withlinear regression r² values≧0.985. Each data point displayed hererepresent the mean±SEM of three or more individual determinations fromthe same well. Each experiment described is representative of findingsfrom three or more independent observations.

In situ Transfection Assay for Migration—Confluent HMEC-1 cells grown on6 well culture plate were transfected with 2.2 μg of expressionplasmids, pSRα ECRTP/DEP-1/3xHA, or pSRα-EphB1/3×HA (Stein et al., GenesDev 12:667-678 (1998)) and circular wounds were prepared at 48 hoursafter transfection as described above. When the wounds were almostclosed (12 h after wounding), monolayers were fixed with 2%paraformaldehyde for 20 min, washed with phosphate buffered saline,permeabilized with 0.02% saponin for 60 min, blocked with 5% goat serumand incubated with 5 □g/mL of monoclonal HA antibody, 12CA5, (BerkeleyAntibody Company (BAbCo), Richmond, Calif.) for 60 min. Coverslips werethen washed with phosphate buffered saline, incubated with biotinylatedgoat anti-mouse IgG (Vector Laboratories, Inc. of Burlingame, Calif.,7.5 μg/mL) for 60 min, washed, incubated with HRP conjugatedavidin-biotin complexes (Vector Laboratories, Inc. of Burlingame,Calif.) for 30 min and finally developed using 6 mg/mL of3,3′-diaminobenzidine (Sigma Chemical Co. of St. Louis, Mo.).

Cornea Pocket Angiogenesis Assay—Agents to be tested for angiogenic oranti-angiogenic activity were immobilized in a slow release form in aninert hydron pellet of approximately 0.2 □l volume, as described Kenyon,Voest, et al. (1996). That pellet is implanted into the cornealepithelium of an anesthetized C57BL mouse in a pocket created bymicro-dissection. Over a 5 to 7 day period angiogenic factors stimulatethe ingrowth of vessels from the adjacent vascularized corneal limbus. Aphotographic record is generated using slit lamp photography. Theappearance, density and extent of these vessels are evaluated andscored. In some cases the time course of the progression is followed inanesthetized animals, prior to sacrifice. Vessels are evaluated forlength, density and the radial surface of the limbus from which theyemanate (expressed as clock-face hours).

Results—Initial experiments were conducted to establish the cell density(cell number/surface area) at which human renal microvascularendothelial cells (HRMEC) display growth arrest in serum supplementedgrowth medium. In situ experiments have shown high level expression ofECRTP/DEP-1 in glomerular and extraglomerular microvascular endothelialcells of human kidney, as well as in arteries and a wide range of othertissues. In FIG. 6A, identical numbers of HRMEC were plated on cellculture plates of 9.6, 28.3, or 78.5 cm², representing 1, 2.9, or 8.1fold the surface area of a 35 mm diameter dish, as indicated. Growthmedium was replaced every 3 days. Depending upon passage number, HRMECreached growth arrest at a density of approximately 1.3-6×10⁴ cells/cm²,a response that supercedes responses to maximal growth stimuli. Doublingtime under density unrestricted conditions is approximately 44 hours.The established human dermal microvascular endothelial cell line,HMEC-1, similarly displayed density-mediated growth arrest properties.

Increasing fibroblast cell density was previously associated withincreases in tyrosine phosphatase activity recovered frommembrane-associated fractions. See e.g., Pallen, C. J. and Tong, P. H.Proc Natl Acad Sci USA 88:6996-7000 (1991). Among membrane-associatedproteins, can surface receptors, including ECRTP/DEP-1, are modified byN-linked glycosylation of the ectodomain region and can be recoveredusing lectin affinity chromatography. Honda et al., Blood 84:4186-4194(1994). Shown in FIG. 6B, tyrosine phosphatase activity of the Triticumvulgaris (wheat germ agglutinin, WGA) lectin fraction recovered fromidentical numbers of HRMEC plated was analyzed for the indicated timesat densities determined by the culture dish surface area. As early as 15hours after plating, marked differences in vanadate-sensitive tyrosinephosphatase activity were evident. Lectin-recovered receptor-associatedtyrosine phosphatase activity was 2 fold higher in cells plated at adensity sufficient to impose growth arrest (8.1×), compared with thoseplated at lower density (1×). As cells plated at lower densities (2.9and 1×) proliferated, increases in activity were seen, eliminating themarked difference. The increased lectin-recovered activity was evidentat times anticipating the imposition of proliferation arrest, suggestingthat either the prevalence of specific tyrosine phosphatases wasincreasing, that the activity of pre-existing phosphatases wasincreased, or that tyrosine phosphatases were being recruited toassociate with lectin recovered proteins. The previous report that DEP-1receptor prevalence increased with increasing cell density lead us toevaluate the activity and distribution of DEP-1. Ostman et al., ProcNatl Acad Sci USA 91:9680-9684 (1994).

Shown in FIG. 7, differences in the amount of immunoprecipitatedECRTP/DEP-1 antigen could not be detected when cells plated for 33 hoursat different densities were compared. Additional experiments failed toshow a change in the ratio of Triton X-100 soluble to insolublefractions at these densities. However, 1.8 fold increases in thevanadate-sensitive ECRTP/DEP-1 associated tyrosine phosphatase activitywere recovered by immunoprecipitation from cells plated at the highest(8.1×) compared with the lowest (1×) cell density. Shown in the lowerpanel immunoblot (FIG. 7), immunoprecipitated ECRTP/DEP-1 is itself atyrosine phosphoprotein in cells pretreated with vanadate. Moreover, thelevel of intrinsic phosphotyrosine is decreased in theimmunoprecipitated ECRTP/DEP-1 recovered from cells plated at highdensity, correlating with the increased tyrosine phosphatase activity inthat fraction. These findings indicate that the abundance of ECRTP/DEP-1does not change acutely in endothelial cells plated at high density, butthat the ECRTP/DEP-1-associated phosphatase activity does increase.Efforts to demonstrate by in gel zymographic phosphatase assays that theincreased activity is intrinsic to ECRTP/DEP-1 have not been successful.

To further pursue the possibility that ECRTP/DEP-1 mediates signalscapable of arresting endothelial proliferation and migration, HMEC-1cells were cotransfected with an expression plasmid driving high levelexpression of an epitope-tagged ECRTP/DEP-1, and with a plasmid drivingexpression of green fluorescent protein to mark transfected cells. Usingadenovirus-assisted transfection method, transfection of 40-50% ofHMEC-1 cells that display survival, migration and proliferationproperties similar to nontransfected cells was routinely accomplished.Shown in FIG. 8A, high level expression of a full length ECRTP/DEP-1imposes marked suppression of BrdU incorporation across a range ofplating densities of transfected cells when compared with the emptyexpression vector.

ECRTP/DEP-1 overexpression imposed similar effects upon endothelialmigration as those observed with proliferation. Shown in FIG. 9A, HMEC-1cells transfected with plasmids driving expression of hemagglutininepitope (HA) tagged versions of either ECRTP/DEP-1/HA or a receptortyrosine kinase, EphB1/HA, were plated at densities to permit them torapidly attain a confluent monolayer. A circular “wound” ofapproximately 500 μm diameter was generated, and migration oftransfected and non-transfected cells to close the “wound” wasdetermined after 33 hours, by staining for the expressed protein HAepitope. Unlike cells transfected with the EphB1/HA control,ECRTP/DEP-1/HA expressing cells did not migrate to contribute to thewound closure. While forced overexpression of ECRTP/DEP-1 can beinformative about the potential for this receptor to affectproliferation or migration, this approach is much less discriminatorythan use of high affinity reagents interacting with endogenouslyexpressed ECRTP/DEP-1s. To this end, a panel of monoclonal antibodiesgenerated against ECRTP/DEP-1 ectodomain sequences was screened foractivity.

Shown in FIG. 8B, bivalent forms of the monoclonal, ECRTPAb1, imposed amarked inhibitory effect on proliferation of HRMEC plated at lowdensity, in spite of repeated growth medium exchanges. Equivalentconcentrations of a class matched monoclonal control antibody wereinactive. Because oligomerization is a critical determinant ofactivation of many receptor tyrosine kinases and phosphatasesWeiss, A.and Schlessinger, J., Cell 94:277-280 (1998)), ECRTPAb1 Fab fragmentswere prepared to test whether bivalency of the interacting monoclonalwas required for activity. Also shown in FIGS. 8B and 8C, equimolarconcentrations of the ECRTPAb1 Fab fragments were inactive as growthinhibitors in endothelial cells plated at subconfluent densities inserum-containing growth medium.

Additional endothelial “wound” closure assays, similar in design tothose presented in FIG. 9A, were conducted to evaluate effects ofbivalent and monovalent ECRTPAb1 on endothelial migration. Displayed inFIG. 9B are the residual fractions of original wound areas remaining atthe times indicated. Phorbol myrisate acetate (PMA) markedly acceleratedthe rate of migration and wound closure, compared with unstimulatedcells in serum-free medium. Bivalent ECRTPAb1 displayed marked activityto inhibit the PMA stimulated migration, while equimolar concentrationsof monovalent Fab fragments, and a control monoclonal were inactive. Thelinear characteristics of time dependent “wound” closure in this assaypermitted us to determine relative migration rates for the population,expressed in FIG. 9C as fractional closure/hr. Effective concentrationsof bivalent ECRTPAb1 (67 and 200 nM) were similar to those active asinhibitors of proliferation (FIG. 8C).

In aggregate, these findings suggested that engagement of endogenousECRTP/DEP-1s by bivalent antibodies can function like a “surrogateligand” to emulate responses normally evoked by an endogenousmembrane-associated ligand upon cell-cell contact. Since the ECRTPAb1Fab fragments were inactive as “surrogate ligands” to inhibit migrationand proliferation in subconfluent cells, it was asked whether they canhave activity as antagonists of endogenous ligand engagement ofECRTP/DEP-1 in cells plated at high density. It was reasoned that Fabfragments can interrupt endogenous ligand-receptor engagement andsubsequent growth arrest signals in cells plated at high density. Shownin FIG. 10, ECRTPAb-1 Fab had a marked effect to release cells from thedensity-imposed inhibition of BrdU uptake that marks S phase entry.

As a final test to determine whether ECRTPAb-1 functions to induce anangiostatic signal, it was tested whether this antibody modifiedangiogenic responses to basic FGF in the mouse corneal pocket assay.Shown in FIG. 11, inclusion of ECRTPAb-1, but not a control IgG, in theimplanted slow release hydron pellet inhibited angiogenesis, scored byreducing the length of capillary sprouts as they approached the sourceof the angiogenic stimulus. This attenuation of capillary length,without effect on radial distribution of new vessels, suggests thatpro-angiogenic basic FGF can diffuse more rapidly from the slow releasepellet than ECRTPAb-1, permitting brisk initiation of angiogenesis withsubsequent attenuation.

Example 3 Method of Screening for Endogenous Ligand of ECRTP/DEP-1

Labeled ECRTP/DEP-1 is used to perform binding studies to identify cellswith ECRTP/DEP-1 ligands using Scatchard analysis; and to performcrosslinking studies which demonstrate the ECRTP/DEP-1 ligand(s) onpolyacrylamide gels. These initial characterization methods are used toidentify cells with high and low numbers of ECRTP/DEP-1 ligand(s) forpurification and isolation studies. Once a cell line with high levels ofECRTP/DEP-1 ligand has been identified, then the protein is purified bythe following approaches:

Approach A: Biochemical Purification

A cell line which expresses high levels of ECRTP/DEP-1 ligand is lysedand the protein from lysates or membrane preparations is purified by gelfiltration followed by purification of the ligand with a columncontaining the ECRTP/DEP-1 bound to a solid phase such as sepharose. Thepurified ligand protein can then be microsequenced and the gene clonedusing degenerate oligonucleotides derived form the protein sequence.

Approach B: cDNA Library Purification

The ECRTP/DEP-1 is radiolabeled with 1251 and then used to screen celllines or tissues by Scratchard analysis for specific binding of ligand.Once such ligand binding is identified, a cDNA library is constructedfrom that tissue or cell line and transfected into a cell line that doesnot exhibit specific binding. These transfected cells are then screenedfor newly acquired specific binding which indicates they have beentransfected with a construct containing the gene for the ECRTP/DEP-1ligand. Plasmid DNA from positive clones is then isolated and sequencedfor identification. A single construct is then transfected back into thenull cells to verify that binding between ligand and receptor ismediated by the transfected gene. Kluzen et al. Proc Natl Acad Sci USA89:4618-4622 (1992).

Alternatively, chimeric ECRTP/DEP-1 and immunoglobulin Fc molecules areconstructed. LaRochelle et al., J Cell Biol 129:357-366 (1995). Thechimeric molecules can then be used to screen for binding to ECRTP/DEP-1ligand on whole cells via flow cytometry. Alternatively, due to thepresence of the immunoglobulin component of the molecule, cell lysatesare screened by immunoblotting or by immunoprecipitation ofmetabolically labeled cells. This technique can identify ECRTP/DEP-1binding proteins by a variety of different methods. Peptide digests ofthe identified proteins are then generated so that peptides can besequenced and the whole molecule cloned by the degenerativeoligonucleotide approach.

Thus, this Example pertains to a method for isolating a ligand for anECRTP/DEP-1, and to a purified and isolated ligand for an ECRTP/DEP-1.The method comprises contacting cells or cell lysates having the ligandor suspected of having the ligand with ECRTP/DEP-1; and isolating theligand which binds with ECRTP/DEP-1. The cells having the ligand areidentified by labeling the ECRTP/DEP-1; screening cell cultures with thelabeled ECRTP/DEP-1; and isolating cells that bind an elevated amount ofthe labeled ECRTP/DEP-1.

The ligand is isolated by lysing the cells and passing the cell lysateover a column containing the ECRTP/DEP-1 bound to a solid phase matrixwithin the column. Alternatively, the ligand is isolated by constructinga cDNA library from the cells binding the ligand; transfecting the cDNAlibrary into a cell line that does not exhibit binding of the ligand;screening the cell line for newly acquired specific binding; isolatingDNA form cells exhibiting specific binding; and sequencing the isolatedDNA to determine the DNA sequence for the ligand.

The ECRTP/DEP-1 is optionally labeled by binding the ECRTP/DEP-1 to animmunoglobulin. In this case, the ligand is isolated byimmunoprecipitation of the ECRTP/DEP-1-ligand-immunoglobulin complex.Alternatively, the ligand of the ECRTP/DEP-1 is isolated using flowcytometry.

Example 4 Identification of Binding Epitope for ECRTPAb-1

A series of 96 eight to nine amino acid peptides was generated. Theeight to nine amino acid peptides span in overlapping epitopes the 351amino acid sequence against which ECRTPAb-1 was derived was generated.These peptides were generated and immobilized in a defined array on thesurface of a membrane that was probed with ECRTPAb-1. Binding ofECRTPAb-1 to peptide #41 in the array was identified using aperoxidase-conjugated anti-mouse IgG second antibody, bychemiluminescence autoradiography (FIG. 12). The sequence of thispeptide derived from the array is n-QSRDTEVL-c (SEQ ID NO: 1). The8-amino acid epitope represents the target sequence within the ECRTPectodomain against which functional ECRTP agonists and antagonistsinteract, based on biological activities of ECRTPAb-1. Antibodies,including humanized antibodies, and other peptides with high affinityfor this defined amino acid sequence have biological activitiescomparable to those demonstrated herein using ECRTPAb-1.

Example 5 Assay for Scoring Biological Activity of Antibodies, Proteinsand Peptides that Bind ECRTP

In an effort to develop a simplified reconstitution assay system capableof scoring biological activities mediated through ECRTP, Chinese HamsterOvary (CHO) cells that do not express endogenous ECRTP were transfectedwith a plasmid construct driving high level expression of ECRTP(pSRα-ECRTP/HA), or catalytically inactive mutated forms(pSRα-ECRTP/HA/C-S, pSRα-ECRTP/HA_(Δ)cy), in these cells. Transientlytransfected CHO cells were then dispersed and plated on 24 well platesand exposed to either control IgG₁ or ECRTPAb-1, or monovalent forms ofECRTP (ECRTPAb-1-Fab). Proliferation of cells transfected with wild typeECRTP, but not with either mutant form, was inhibited (approximately40%) by incubation with ECRTPAb-1, but not with either control IgG₁ orECRTPAb-1-Fab.

In parallel, ECRTP was immunoprecipitated from CHO cells transfectedwith these plasmids and expressing equivalent amounts of ECRTP or mutantforms (shown in the inserts above FIGS. 13A and 13B) following a timecourse of exposure (0-30 min) to either ECRTPAb-1 or ECRTPAb-1-Fab.Shown in the immunoblot panels on the right in FIGS. 13A and 13B, Ab1evoked a rapid de-phosphorylation of tyrosine residues on the wild type(WT) ECRTP, but had no effect on catalytically inactive C/S mutantforms. These findings provide primary evidence that the catalyticphosphatase function is critical to the action of ECRTPAb-1 to inhibitproliferation and to provoke de-phosphorylation of ECRTP.

Finally, co-expression of either catalytically inactive C/S or Cydeleted forms of ECRTP with the functional ECRTP form abrogatesinhibitory effects of the ECRTPAb-1 on CHO proliferation (FIG. 13B).This proliferation assay thus evaluates whether putativecounter-receptors (ligands) are competent to function through ECRTP toinhibit cell growth. In each case, the monovalent and bivalent forms ofECRTPAb-1 provide a reagent that can be used in the assay to definefunctional counter-receptors.

Example 6 Transgenic Animal Having Catalytically Inactive ECRTP/DEP-1

A transgenic mouse expressing a catalytically inactive form ofECRTP/DEP-1 was prepared. The mouse was prepared using a “knockout”approach with respect to the ECRTP/DEP-1 gene. Embryonic death wasobserved in homozygous “knockout” animals. Heterozygous animalsdisplayed significant vascularization malformations. Thus, thetransgenic mouse further established the role of ECRTP/DEP-1 in cellgrowth, cell survival and angiogenesis.

REFERENCES

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

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It will be understood that various details of the invention can bechanged without departing from the scope of the invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation—the invention being defined by theclaims.

1-14. (canceled)
 15. A pharmaceutical composition comprising an isolatedand purified biologically active ECRTP/DEP-1 polypeptide, or amide,conjugated, cyclized, fragment, chemically modified embodiment thereof,and a pharmaceutically acceptable carrier.
 16. The pharmaceuticalcomposition of claim 15, wherein the polypeptide further comprises anectodomain of the ECRTP/DEP-1.
 17. The pharmaceutical composition ofclaim 16, wherein the polypeptide further comprises an eight amino acidepitope of the ectodomain of the ECRTP/DEP-1, the epitope having thesequence QSRDTEVL, or an eight amino acid epitope of the ectodomain ofthe ECRTP/DEP-1 having an analog of the sequence QSRDTEVL.
 18. Thepharmaceutical composition of claim 15, further comprising a cellexpressing the ECRTP/DEP-1 polypeptide.
 19. A method of screening acandidate substance for an ability to modulate a receptor tyrosinephosphatase, the method comprising: (a) establishing a test samplecomprising a receptor tyrosine phosphatase; (b) administering acandidate substance to the test sample; and (c) measuring a receptortyrosine phosphatase biological activity in the test sample; (d)detecting phosphotyrosine residues on the receptor tyrosine phosphatase;and (e) determining that the candidate substance modulates the receptortyrosine phosphatase if the receptor tyrosine phosphatase biologicalactivity measured for the test sample is greater or less than thereceptor tyrosine phosphatase biological activity measured for a controlsample and if the amount of phosphotyrosine residues on the receptortyrosine phosphatase is greater or less than an amount ofphosphotyrosine residues on a receptor tyrosine phosphate derived from acontrol sample.
 20. The method of claim 19, wherein the test and controlsamples further comprise a cell, and the receptor tyrosine phosphataseis expressed in the cell.
 21. The method of claim 20, wherein the testand control sample comprise cells expressing an ECRTP/DEP-1.
 22. Themethod of claim 21, wherein the ECRTP/DEP-1 activity is selected fromthe group consisting of modulation of endothelial cell migration andproliferation, modulation of density induced growth arrest, modulationof angiogenesis and combinations thereof.
 23. The method of claim 19,wherein the candidate substance further comprises a cell or cell lysatecomprising a natural ligand for the receptor tyrosine phosphatase, andthe method further comprises isolating the natural ligand for thereceptor tyrosine phosphatase.
 24. The method of claim 23, wherein thereceptor tyrosine phosphatase comprises the ECRTP/DEP-1.
 25. The methodof claim 24, wherein the ligand is isolated by lysing the cells andpassing the cell lysate over a column containing the ECRTP/DEP-1 boundto a solid phase matrix within the column.
 26. The method of claim 24,wherein the ligand is isolated by constructing a cDNA library from thecells binding the ligand; transfecting the cDNA library into a cell linethat does not exhibit binding of the ligand; screening the cell line fornewly acquired specific binding; isolating DNA form cells exhibitingspecific binding; and sequencing the isolated DNA to determine the DNAsequence for the ligand.
 27. A recombinant cell line suitable for use inthe assay of claim
 19. 28. A method of screening a candidate substancefor an ability to modulate ECRTP/DEP-1 biological activity, the methodcomprising: (a) establishing a test sample comprising an ECRTP/DEP-1polypeptide or fragment thereof; (b) administering a candidate substanceto the test sample; and (c) measuring an interaction, effect, orcombination thereof, of the candidate substance on the test sample tothereby determine the ability of the candidate substance to modulateECRTP/DEP-1 biological activity.
 29. The method of claim 28, wherein thetest sample further comprises a cell expressing ECRTP/DEP-1, and whereinthe step of measuring an interaction, effect, or combination thereof, ofthe candidate substance on the test sample further comprises: (i)comparing the interaction, effect, or combination thereof, of thecandidate substance on the test sample with the interaction, effect, orcombination thereof, of the candidate substance on a cell not expressingECRTP/DEP-1; and (ii) determining that candidate compound modulatesECRTP/DEP-1 activity by demonstrate a lack of interaction, effect orcombination thereof, of the candidate compound on cells not expressingECRTP/DEP-1.
 30. The method of claim 28, wherein said step of measuringan interaction, effect, or combination thereof, of the candidatesubstance on the test sample further comprises measuring binding betweenthe candidate substance and the test sample by: (i) contacting thecandidate substance with an ECRTP/DEP-1 polypeptide or fragment thereofunder conditions favorable to binding the candidate with an ECRTP/DEP-1polypeptide or fragment thereof to form a complex therebetween; and (ii)detecting the complex.
 31. The method of claim 30, wherein the complexis detected via a label conjugated to the ECRTP/DEP-1 polypeptide orfragment thereof; via a labeled reagent that specifically binds to thecomplex subsequent to its formation; or via a competition assay with asubstance known to bind the ECRTP/DEP-1 polypeptide for fragmentthereof.
 32. The method of claim 30, wherein the ECRTP/DEP-1 polypeptideor fragment thereof is conjugated with a detectable label.
 33. Themethod of claim 32, wherein the step of detecting the complex furthercomprises: (i) separating the complex from unbound labeled ECRTP/DEP-1polypeptide or fragment thereof; and (ii) detecting the detectable labelwhich is present in the complex or which is unbound.
 34. The method ofclaim 30, wherein the ECRTP/DEP-1 polypeptide fragment is a ECRTP/DEP-1ectodomain fragment.
 35. The method of claim 34, wherein the ECRTP/DEP-1ectodomain fragment comprises an eight amino acid epitope having thesequence n-QSRDTEVL-c.
 36. The method of claim 30, wherein the candidatesubstance is an antibody, or derivative or fragment thereof.
 37. Themethod of claim 36, wherein the candidate antibody, or derivative orfragment thereof, is derived from a recombinant phage-displayed antibodylibrary.
 38. A kit for use screening a candidate substance for anability to modulate ECRTP/DEP-1 biological activity, the kit comprisinga ECRTP/DEP-1 ectodomain polypeptide, or fragment thereof, contained ina first container.
 39. The kit of claim 38, wherein the ECRTP/DEP-1ectodomain polypeptide, or fragment thereof, comprises an eight aminoacid epitope having the sequence n-QSRDTEVL-c.
 40. The kit of claim 38,further comprising a solid phase support.
 41. The kit of claim 40, wherethe ECRTP/DEP-1 ectodomain polypeptide, or fragment thereof, isimmobilized to the solid phase support.
 42. The kit of claim 38, furthercomprising a detectable label.
 43. The kit of claim 41, wherein thedetectable label is contained in another container or whereinECRTP/DEP-1 ectodomain polypeptide, or fragment thereof, comprises thedetectable label.
 44. The kit of claim 43, wherein the detectable labelis a radioactive label or an enzyme.
 45. An isolated antibody, or afragment or derivative thereof, which specifically binds to an epitopepresent within amino acids 175-536 of a human ECRTP/DEP-1 polypeptide,wherein the human ECRTP/DEP-1 polypeptide comprises an amino acidsequence as set forth in SEQ ID NO:
 4. 46. The isolated antibodyfragment of claim 45, wherein the isolated antibody fragment is selectedfrom the group consisting of an Fab fragment, an Fab′ fragment, anF(ab′)₂ fragment, an F(v) fragment, and an single chain fragmentvariable (scFv) fragment.
 47. The isolated antibody, or the fragment orderivative thereof, of claim 45, wherein the isolated antibody comprisesa monoclonal antibody, or a fragment or derivative thereof.
 48. Theisolated antibody, or the fragment or derivative thereof, of claim 47,wherein the isolated antibody is humanized.
 49. The isolated antibody,or the fragment or derivative thereof, of claim 45, which binds an eightamino acid epitope consisting of the sequence QSRDTEVL (SEQ ID NO: 1).50. The isolated antibody, or the fragment or derivative thereof, ofclaim 49, wherein the isolated antibody, or the fragment or derivativethereof, is a monoclonal antibody, or a fragment or derivative thereof.51. The isolated antibody, or the fragment or derivative thereof, ofclaim 50, wherein the antibody, or the fragment or derivative thereof,is human or humanized.
 52. The isolated antibody, or the fragment orderivative thereof, of claim 45, in a pharmaceutically acceptablediluent or excipient.
 53. The isolated antibody, or the fragment orderivative thereof, of claim 52, wherein the pharmaceutically acceptablediluent or excipient is pharmaceutically acceptable for use in humans.54. An isolated antibody, or a fragment or derivative thereof, whichspecifically binds to an epitope of an ECRTP/DEP-1 polypeptideextracellular domain, wherein the ECRTP/DEP-1 polypeptide extracellulardomain comprises amino acids 175-536 of SEQ ID NO: 4 and the epitopecomprises SEQ ID NO:
 1. 55. The isolated antibody fragment of claim 54,wherein the isolated antibody fragment is selected from the groupconsisting of an Fab fragment, an Fab′ fragment, an F(ab′)₂ fragment, anF(v) fragment, and an single chain fragment variable (scFv) fragment.56. The isolated antibody, or the fragment or derivative thereof, ofclaim 54, wherein the isolated antibody is humanized.
 57. The isolatedantibody, or the fragment or derivative thereof, of claim 54, in apharmaceutically acceptable diluent or excipient.
 58. The isolatedantibody, or the fragment or derivative thereof, of claim 57, whereinthe pharmaceutically acceptable diluent or excipient is pharmaceuticallyacceptable for use in humans.
 59. An isolated antibody, or a fragment orderivative thereof, which specifically binds an extracellular domain ofan ECRTP/DEP-1 polypeptide comprising an amino acid sequence as setforth in SEQ ID NO: 4 and wherein the antibody, fragment, or derivativethereof has activity in modulating angiogenesis.
 60. The isolatedantibody, or the fragment or derivative thereof, of claim 59, or afragment or derivative thereof, wherein the isolated antibody, fragment,or derivative thereof has activity in modulating angiogenesis in anassay selected from the group consisting of a planar endothelialmigration assay, an in situ transfection assay for migration, a corneapocket angiogenesis assay, a chick chorioallantoic membrane assay, aproliferation assay, and an endothelial wound closure assay.
 61. Theantibody fragment, or the fragment or derivative thereof, of claim 59,wherein the antibody fragment is selected from the group consisting ofan Fab fragment, an Fab′ fragment, an F(ab′)₂ fragment, an F(v)fragment, and an single chain fragment variable (scFv) fragment.
 62. Theisolated antibody, or the fragment or derivative thereof, of claim 59,wherein the isolated antibody is a monoclonal antibody, or a fragment orderivative thereof.
 63. The isolated antibody, or the fragment orderivative thereof, of claim 62, wherein the isolated antibody ishumanized.
 64. The isolated antibody, or the fragment or derivativethereof, of claim 59, in a pharmaceutically acceptable diluent orexcipient.
 65. The isolated antibody, or the fragment or derivativethereof, of claim 64, wherein the pharmaceutically acceptable diluent orexcipient is pharmaceutically acceptable for use in humans.
 66. Theisolated antibody, or the fragment or derivative thereof, of claim 59,wherein the isolated antibody has a binding specificity of a monoclonalantibody produced by a hybridoma cell line having American Type CultureCollection (ATCC) accession number HB12570.
 67. The isolated antibody,or the fragment or derivative thereof, of claim 59, or a fragment orderivative thereof, wherein the activity in modulating angiogenesis isinhibition of angiogenesis.
 68. An isolated antibody, or a fragment orderivative thereof, which specifically binds an epitope present withinamino acids 175-536 of a human ECRTP/DEP-1 polypeptide comprising anamino acid sequence as set forth in SEQ ID NO: 4, wherein the isolatedantibody, or the fragment or derivative thereof, has activity inmodulating angiogenesis.
 69. The isolated antibody, or the fragment orderivative thereof, of claim 68, wherein the isolated antibody, or thefragment or derivative thereof, has activity in modulating angiogenesisin an assay selected from the group consisting of a planar endothelialmigration assay, an in situ transfection assay for migration, a corneapocket angiogenesis assay, a chick chorioallantoic membrane assay, aproliferation assay, and an endothelial wound closure assay.
 70. Theisolated antibody fragment of claim 68, wherein the isolated antibodyfragment is selected from the group consisting of an Fab fragment, anFab′ fragment, an F(ab′)₂ fragment, an F(v) fragment, and an singlechain fragment variable (scFv) fragment.
 71. The isolated antibody, orthe fragment or derivative thereof, of claim 68, wherein the isolatedantibody is a monoclonal antibody, or a fragment or derivative thereof.72. The isolated antibody, or the fragment or derivative thereof, ofclaim 71, wherein the isolated antibody is humanized.
 73. The isolatedantibody, or the fragment or derivative thereof, of claim 68, in apharmaceutically acceptable diluent or excipient.
 74. The isolatedantibody, or the fragment or derivative thereof, of claim 73, whereinthe pharmaceutically acceptable diluent or excipient is pharmaceuticallyacceptable for use in humans.
 75. The isolated antibody, or the fragmentor derivative thereof, of claim 68, or a fragment or derivative thereof,wherein the activity in modulating angiogenesis is inhibition ofangiogenesis.
 76. An isolated antibody, or a fragment or derivativethereof, which specifically binds to an epitope of an ECRTP/DEP-1polypeptide extracellular domain, the epitope comprising SEQ ID NO: 1,wherein the antibody, fragment, or derivative thereof has activity inmodulating angiogenesis.
 77. The isolated antibody, or the fragment orderivative thereof, of claim 76, wherein the antibody, fragment, orderivative thereof has activity in modulating angiogenesis in an assayselected from the group consisting of a planar endothelial migrationassay, an in situ transfection assay for migration, a cornea pocketangiogenesis assay, a chick chorioallantoic membrane assay, aproliferation assay, and an endothelial wound closure assay.
 78. Theantibody fragment of claim 77, wherein the antibody fragment is selectedfrom the group consisting of an Fab fragment, an Fab′ fragment, anF(ab′)₂ fragment, an F(v) fragment, and an single chain fragmentvariable (scFv) fragment.
 79. The isolated antibody, or the fragment orderivative thereof, of claim 76, wherein the isolated antibody is amonoclonal antibody or a fragment or derivative thereof.
 80. Theisolated antibody, or the fragment or derivative thereof, of claim 79,wherein the antibody is humanized.
 81. The isolated antibody, or thefragment or derivative thereof, of claim 76, in a pharmaceuticallyacceptable diluent or excipient.
 82. The isolated antibody, or thefragment or derivative thereof, of claim 81, wherein thepharmaceutically acceptable diluent or excipient is pharmaceuticallyacceptable for use in humans.
 83. The isolated antibody, or the fragmentor derivative thereof, of claim 76, or a fragment or derivative thereof,wherein the activity in modulating angiogenesis is inhibition ofangiogenesis.
 84. A composition for modulating angiogenesis, thecomposition comprising: (a) a therapeutically effective amount of anisolated antibody, or a fragment or derivative thereof, which binds toan eight amino acid epitope consisting of SEQ ID NO: 1 present within ahuman ECRTP/DEP-1 density enhanced phosphatase-1 polypeptide comprisingan amino acid sequence as set forth in SEQ ID NO: 4; and (b) a diluentor excipient pharmaceutically acceptable in humans.
 85. The compositionof claim 84, wherein the antibody, or the fragment or derivativethereof, is human or humanized.